Oxime conjugates and methods for their formation and use

ABSTRACT

The present invention relates to biodegradable biocompatible polyketals, methods for their preparation, and methods for creating animals by administration of biodegradable biocompatible polyketals. In one aspect, a method for forming the biodegradable biocompatible polyketals comprises combining a glycol-specific oxidizing agent with a polysaccharide to form an aldehyde intermediate, which is combined with a reducing agent to form the biodegradable biocompatible polyketal. The resultant biodegradable biocompatible polyketals can be chemically modified to incorporate additional hydrophilic moieties. A method for treating animals includes the administration of the biodegradable biocompatible polyketal in which biologically active compounds or diagnostic labels can be disposed. The present invention also relates to chiral polyketals, methods for their preparation, and methods for use in chromatographic applications, specifically in chiral separations. A method for forming the chiral polyketals comprises combining a glycol-specific oxidizing agent with a polysaccharide to form an aldehyde intermediate, which is combined with a suitable reagent to form the chiral polyketal. A method for use in chiral separations includes the incorporation of the chiral polyketals in the mobile phase during a chromatographic separation, or into chiral stationary phases such as gels. The present invention further relates to chiral polyketals as a source for chiral compounds, and methods for generating such chiral compounds.

PRIORITY CLAIM

The present application claims the benefit under 35 U.S.C. 371 ofInternational Application No.: PCT/US03/33584 (published PCT applicationNo. WO 04/09082), filed Jul. 18, 2003, which claims priority to U.S.Patent Application No. 60/397,283, filed Jul. 19, 2002, the entirecontents of each of the above cited applications are incorporated hereinby reference.

GOVERNMENT FUNDING

The present invention was made with support, in part, from a grant fromthe National Center for Research Resources of the National Institutes ofHealth (Number R21-RR14221) and a DoE grant (Number DE-FG02-00ER63057).Accordingly, the United States Government may have certain rights inthis invention.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by any-one of the patentdocument or the patent disclosure, as it appears in the Patent andTrademark Office patent file or records, but otherwise reserves allcopyright rights whatsoever.

BACKGROUND OF THE INVENTION

Traditionally, pharmaceuticals have primarily consisted of smallmolecules that are dispensed orally (as solid pills and liquids) or asinjectables. Over the past three decades, however, sustained releaseformulations (i.e., compositions that control the rate of drug deliveryand allows delivery of the therapeutic agent at the site where it isneeded) have become increasingly common and complex. Nevertheless, manyquestions and challenges regarding the development of new treatments aswell as the mechanisms with which to administer them remain to beaddressed.

Although considerable research efforts in this area have led tosignificant advances, drug delivery methods/systems that have beendeveloped over the years and are currently used, still exhibit specificproblems that require some investigating. For example, many drugsexhibit limited or otherwise reduced potencies and therapeutic effectsbecause of they are generally subject to partial degradation before theyreach a desired target in the body. Once administered, sustained releasemedications deliver treatment continuously, e.g. for days or weeks,rather than for a short period of time (hours or minutes). Furthermore,orally administered therapeutics are generally preferable overinjectable medications, which are often more expensive and are morechallenging to administer, and thus it would be highly desirable ifinjectable medications could simply be dosed orally. However, this goalcannot be achieved until methods are developed to safely shepherd drugsthrough tissue barriers, such as epithelial or dermal barriers, orspecific areas of the body, such as the stomach, where low pH candegrade or destroy a medication, or through an area where healthy tissuemight be adversely affected.

One objective in the field of drug delivery systems, therefore, is todeliver medications intact to specifically targeted areas of the bodythrough a system that can control the rate and time of administration ofthe therapeutic agent by means of either a physiological or chemicaltrigger. Over the past decade, materials such as polymeric microspheres,polymer micelles, soluble polymers and hydrogel-type materials have beenshown to be effective in enhancing drug targeting specificity, loweringsystemic drug toxicity, improving treatment absorption rates, andproviding protection for pharmaceuticals against biochemicaldegradation, and thus have shown great potential for use in biomedicalapplications, particularly as components of drug delivery devices.

The design and engineering of biomedical polymers (e.g., polymers foruse under physiological conditions) are generally subject to specificand stringent requirements. In particular, such polymeric materials mustbe compatible with the biological milieu in which they will be used,which often means that they show certain characteristics ofhydrophilicity. They also have to demonstrate adequate biodegradability(i.e., they degrade to low molecular weight species. The polymerfragments are in turn metabolized in the body or excreted, leaving notrace).

Biodegradability is typically accomplished by synthesizing or usingpolymers that have hydrolytically unstable linkages in the backbone. Themost common chemical functional groups with this characteristic areesters, anhydrides, orthoesters, and amides. Chemical hydrolysis of thehydrolytically unstable backbone is the prevailing mechanism for thepolymer's degradation. Biodegradable polymers can be either natural orsynthetic. Synthetic polymers commonly used in medical applications andbiomedical research include polyethyleneglycol (pharmacokinetics andimmune response modifier), polyvinyl alcohol (drug carrier), andpoly(hydroxypropylmetacrylamide) (drug carrier). In addition, naturalpolymers are also used in biomedical applications. For instance,dextran, hydroxyethylstarch, albumin and partially hydrolyzed proteinsfind use in applications ranging from plasma substitute, toradiopharmaceutical to parenteral nutrition. In general, syntheticpolymers may offer greater advantages than natural materials in thatthey can be tailored to give a wider range of properties and morepredictable lot-to-lot uniformity than can materials from naturalsources. Synthetic polymers also represent a more reliable source of rawmaterials, one free from concerns of infection or immunogenicity.Methods of preparing polymeric materials are well known in the art.However, synthetic methods that successfully lead to the preparation ofpolymeric materials that exhibit adequate biodegradability,biocompatibility, hydrophilicity and minimal toxicity for biomedical useare scarce. The restricted number and variety of biopolymers currentlyavailable attest to this.

Therefore a need exists in the biomedical field for low-toxicity,biodegradable, biocompatible, hydrophilic polymer conjugates comprisingpharmaceutically useful modifiers, which overcome or minimize theabove-referenced problems. Such polymer conjugates would find use inseveral applications, including components for biomedical preparations,pharmaceutical formulations, medical devices, implants, and thepackaging/delivery of therapeutic, diagnostic and prophylatic agents.

SUMMARY OF THE INVENTION

The present invention discloses a polymer conjugate that isbiodegradable, biocompatible and exhibits little toxicity and/orbioadhesivity in vivo, and contains one or more modifiers covalentlyattached to the polymer via oxime-containing linkages.

In one aspect, the invention encompasses a conjugate comprising acarrier substituted with one or more occurrences of a moiety having thestructure:

-   -   wherein each occurrence of M is independently a modifier; and    -   each occurrence of L^(M) is independently an oxime-containing        linker.

In certain embodiments, each occurrence of L^(M) is independently amoiety having the structure:

-   -   wherein each occurrence of L^(M1) is independently a substituted        or unsubstituted, cyclic or acyclic, linear or branched        C₀₋₁₂alkylidene or CO₁₂ alkenylidene moiety wherein up to two        non-adjacent methylene units are independently optionally        replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),        NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,        NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1),        NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein each occurrence of        R^(Z1) and R^(Z2) is independently hydrogen, alkyl, heteroalkyl,        aryl, heteroaryl or acyl.

In certain other embodiments, one or more occurrences of L^(M1)independently comprises a maleimide- or N-hydroxysuccinimideester-containing crosslinker. In yet other embodiments, one or moreoccurrences of L^(M1) independently comprises a4-(N-maleimidomethyl)cyclohexane-1-carboxylate, m-maleimidobenzoyl or a4-(p-maleimidophenyl)butyrate crosslinker.

In certain other embodiment, one or more occurrences of M comprises, oris attached to the carrier through, a biodegradable bond. In certainexemplary embodiments, the biodegradable bond is selected from the groupconsisting of acetal, ketal, amide, ester, thioester, enamine, imine,imide, dithio, and phosphoester bond.

In certain other embodiments, the carrier is a hydrophilic biodegradablepolymer selected from the group consisting of carbohydrates,glycopolysaccharides, glycolipids, glycoconjugates, polyacetals,polyketals, and derivatives thereof. In certain exemplary embodiments,the carrier is a naturally occurring linear and branched biodegradablebiocompatible homopolysaccharide selected from the group consisting ofcellulose, amylose, dextran, levan, fucoidan, carraginan, inulin,pectin, amylopectin, glycogen and lixenan. In certain other exemplaryembodiments, the carrier is a naturally occurring linear and branchedbiodegradable biocompatible heteropolysaccharide selected from the groupconsisting of agarose, hyluronan, chondroitinsulfate, dermatansulfate,keratansulfate, alginic acid and heparin. In yet other exemplaryembodiments, the carrier is a hydrophilic polymer selected from thegroup consisting of polyacrylates, polyvinyl polymers, polyesters,polyorthoesters, polyamides, polypeptides, and derivatives thereof. Instill other exemplary embodiments, the carrier is a biodegradablebiocompatible polyacetal wherein at least a subset of the polyacetalrepeat structural units have the following chemical structure:

-   -   wherein for each occurrence of the n bracketed structure, one of        R¹ and R² is hydrogen, and the other is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation. In further exemplary embodiments, the        carrier is a biodegradable biocompatible polyketal wherein at        least a subset of the polyketal repeat structural units have the        following chemical structure:

-   -   wherein each occurrence of R¹ and R¹ is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R¹, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation.

In other embodiments, in the conjugates of the invention, one or moreoccurrences of M comprise a biologically active modifier. In certainexemplary embodiments, one or more occurrence of M is selected from thegroup consisting of proteins, antibodies, antibody fragments, peptides,antineoplastic drugs, hormones, cytokines, enzymes, enzyme substrates,receptor ligands, lipids, nucleotides, nucleosides, metal complexes,cations, anions, amines, heterocycles, heterocyclic amines, aromaticgroups, aliphatic groups, intercalators, antibiotics, antigens,immunomodulators, and antiviral compounds.

In certain other embodiments, one or more occurrence of M comprises adetectable label. In certain exemplary embodiments, one or moreoccurrence of M comprises atoms or groups of atoms comprisingradioactive, paramagnetic, superparamagnetic, fluorescent, or lightabsorbing structural domains.

In certain other embodiments, one or more occurrences of M comprise adiagnostic label. In certain exemplary embodiments, one or moreoccurrence of M comprises radiopharmaceutical or radioactive isotopesfor gamma scintigraphy and PET, contrast agent for Magnetic ResonanceImaging (MRI), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves, microwaves and/or fluorophores.

In certain embodiments, the conjugates of the invention arewater-soluble. In certain exemplary embodiments, the inventive conjugatecomprises a biologically active modifier and a detectable label.

In certain embodiments, the carrier is a linear macromolecule, abranched macromolecule, a globular macromolecule, a graft copolymer, acomb copolymer, a nanoparticle or a lipid-based carrier. In certainexemplary embodiments, the lipid-based carrier is a liposome.

In another aspect, the invention encompasses compounds having thestructure R^(N1)R^(N2)N—O-L¹; wherein R¹ and R^(N2) are independentlyhydrogen, an aliphatic, alicyclic, heteroaliphatic, heterocyclic, arylor heteroaryl moiety, or a nitrogen protecting group, or R^(N1) andR^(N2), taken together with the nitrogen atom to which they areattached, form a substituted or unsubstituted heterocyclic or heteroarylmoiety; and L¹ is an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aryl or heteroaryl moiety comprising a functional groupadapted for covalent binding to a modifier. In certain exemplaryembodiments, L¹ is a moiety having the structure—(CR^(L1)R^(L2))_(p)-Q-, wherein p is an integer from 0-6, R^(L1) andR^(L2) are independently hydrogen, an aliphatic, alicyclic,heteroaliphatic, heterocyclic, aryl or heteroaryl moiety or WR^(W1)wherein W is O, S, NH, CO, SO₂, COO, CONH, and R^(W1) is hydrogen, analiphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl,alkylaryl or alkylheteroaryl moiety, and Q is a moiety comprising afunctional group adapted for covalent binding to a modifier. In certainother exemplary embodiments, L¹ is —CH₂)_(p) wherein p is an integerfrom 0-5, and Q is a succinimidyl ester moiety having the structure:

In yet other exemplary embodiments, L¹ is —(CH₂)_(p1)—CH(OH)CH₂NH—wherein p₁ is an integer from 1-5, and Q is a maleimidyl moiety havingthe structure:

-   -   wherein p₂ is an integer from 1-5.

In certain exemplary embodiments, in the compounds of the invention,R^(N1)R^(N2)N— is a moiety having the structure:

In another aspect, the invention provides compounds having thestructure:

-   -   wherein M is a modifier; L^(M1) is a substituted or        unsubstituted, cyclic or acyclic, linear or branched        C₀₋₁₂alkylidene or C₀₋₁₂alkenylidene moiety wherein up to two        non-adjacent methylene units are independently optionally        replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),        NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,        NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1),        NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein each occurrence of        R^(Z1) and R^(Z2) is independently hydrogen, alkyl, heteroalkyl,        aryl, heteroaryl or acyl; and R^(N1) and R^(N2) are        independently hydrogen, an aliphatic, alicyclic,        heteroaliphatic, heterocyclic, aryl or heteroaryl moiety, or a        nitrogen protecting group, or R^(N1) and R^(N2), taken together        with the nitrogen atom to which they are attached, form a        substituted or unsubstituted heterocyclic or heteroaryl moiety.

In certain exemplary embodiments, L^(M1) comprises an NHS estercrosslinker and the compound has the structure:

-   -   wherein p is 0-5.

In certain other exemplary embodiments, L¹ comprises a maleimidecrosslinker and the compound has the structure:

-   -   wherein p₁ and p₂ are independently integers from 1-5.

In certain exemplary embodiments, in the compounds having the structure:

R^(N1)R^(N2)N— is a moiety having the structure:

In another aspect, the invention provides a method for preparing aconjugate comprising a carrier substituted with one or more occurrencesof a moiety having the structure:

-   -   wherein each occurrence of M is independently a modifier; and    -   each occurrence of L^(M) is independently an oxime-containing        linker;    -   said method comprising steps of:    -   providing a carrier;    -   providing one or more modifiers;    -   providing one or more compounds having the structure:        R^(N1)R^(N2)N—O-L¹; wherein R^(N1) and R^(N2) are independently        hydrogen, an aliphatic, alicyclic, heteroaliphatic,        heterocyclic, aryl or heteroaryl moiety, or a nitrogen        protecting group, or R^(N1) and R^(N2), taken together, form a        substituted or unsubstituted alicyclic, aryl or heteroaryl        moiety; and each occurrence of L¹ is independently an aliphatic,        alicyclic, heteroaliphatic, heterocyclic, aryl or heteroaryl        moiety comprising a functional group adapted for covalent        binding to the modifier; and    -   reacting the one or more compounds of structure        R^(N1)R^(N2)N—O-L¹ with the carrier and the one or more        modifiers under suitable conditions so that at least one        —O—NR^(N1)R^(N2) moiety is covalently attached to the carrier        via an oxime linkage, thereby generating the conjugate.

In another aspect, the invention provides a method for preparing aconjugate comprising a carrier substituted with one or more occurrencesof a moiety having the structure:

-   -   wherein each occurrence of M is independently a modifier; and    -   each occurrence of L^(M) is independently an oxime-containing        linker;    -   said method comprising steps of:    -   providing a carrier;    -   providing one or more compounds having the structure:

-   -   wherein L^(M1) is a substituted or unsubstituted, cyclic or        acyclic, linear or branched C₀₋₁₂alkylidene or C₀₋₁₂alkenylidene        moiety wherein up to two nonadjacent methylene units are        independently optionally replaced by CO, CO₂, COCO, CONR^(Z1),        OCONR^(Z1), NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO,        NR^(Z1)CO₂, NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1),        NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein each occurrence of        R^(Z1) and R^(Z2) is independently hydrogen, alkyl, heteroalkyl,        aryl, heteroaryl or acyl; and R^(N1) and R^(N2) are        independently hydrogen, an aliphatic, alicyclic,        heteroaliphatic, heterocyclic, aryl or heteroaryl moiety, or a        nitrogen protecting group, or R^(N1) and R^(N2), taken together        with the nitrogen atom to which they are attached, form a        substituted or unsubstituted heterocyclic or heteroaryl moiety;        and    -   reacting the carrier with the one or more compounds of        structure:

-   -   under suitable conditions so that at least one —O—NR^(N1)R^(N2)        moiety is covalently attached to the carrier via an oxime        linkage, thereby generating the conjugate.

In certain exemplary embodiments, R^(N1) and R^(N2) are each hydrogen.In certain exemplary embodiments, in the one or more compounds ofstructure R^(N1)R^(N2)N—O-L¹; at least one of R^(N1) and R^(N2) is anitrogen protecting group; and the method further comprises the step ofhydrolyzing the one or more compounds having the structureR^(N1)R^(N2)N—O-L¹ to form one or more compounds having the structureH₂N—O-L¹ prior to reacting with the carrier. In certain exemplaryembodiments, in the one or more compounds of structureR^(N1)R^(N2)N—O-L¹, R^(N1)R^(N2)N— has the structure CH₃CH₂OC(CH₃)═N—;and the one or more compounds have the following structure:

In certain exemplary embodiments, in the one or more compounds ofstructure

at least one of R^(N1) and R^(N2) is a nitrogen protecting group; andthe method further comprises the step of hydrolyzing the one or morecompounds having the structure:

-   -   to form one or more compounds having the structure:

-   -   prior to reacting with the carrier.

In certain exemplary embodiments, in the one or more compounds ofstructure:

R^(N1)R^(N2)N— has the structure CH₃CH₂OC(CH₃)═N—; and the one or morecompounds have the following structure:

In certain exemplary embodiments, in practicing the method of theinvention, the carrier is a biodegradable biocompatible polyacetalwherein at least a subset of the polyacetal repeat structural units havethe following chemical structure:

-   -   wherein for each occurrence of the n bracketed structure, one of        R¹ and R² is hydrogen, and the other is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation. In certain embodiments, the carbonyl group        is an aldehyde.

In certain exemplary embodiments, in practicing the method of theinvention, the carrier is a biodegradable biocompatible polyketalwherein at least a subset of the polyketal repeat structural units havethe following chemical structure:

-   -   wherein each occurrence of R¹ and R² is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation.

In another aspect, the invention provides compositions comprising theconjugate of the invention and a pharmaceutically suitable carrier ordiluent. In certain embodiments, the inventive compositions comprise aconjugate associated with an effective amount of a therapeutic agent;wherein the therapeutic agent is incorporated into an released from saidconjugate matrix by degradation of the conjugate matrix or diffusion ofthe agent out of the matrix over a period of time. In certainembodiments, the conjugate is further associated with a diagnosticagent.

In yet another aspect, the invention provides a method of administeringto a patient in need of treatment, comprising administering to thesubject an effective amount of a suitable therapeutic agent; whereinsaid therapeutic agent is associated with and released from a conjugateof the invention by degradation of the conjugate matrix or diffusion ofthe agent out of the matrix over a period of time. In certainembodiments, the therapeutic agent is locally delivered by implantationof said conjugate matrix incorporating the therapeutic agent. In certainembodiments, the therapeutic agent is selected from the group consistingof: vitamins, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents.

In certain other exemplary embodiments, the method further comprisesadministering with the therapeutic agent additional biologically activecompounds selected from the group consisting of vitamins, anti-AIDSsubstances, anti-cancer substances, antibiotics, immunosuppressants,anti-viral substances, enzyme inhibitors, neurotoxins, opioids,hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants,muscle relaxants and anti-Parkinson substances, anti-spasmodics andmuscle contractants including channel blockers, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, vasodilating agents, inhibitors of DNA, RNA or proteinsynthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal andnon-steroidal anti-inflammatory agents, anti-angiogenic factors,anti-secretory factors, anticoagulants and/or antithrombotic agents,local anesthetics, ophthalmics, prostaglandins, anti-depressants,anti-psychotic substances, anti-emetics, imaging agents, and combinationthereof.

In certain embodiments, in practicing the method of the invention, theconjugate further comprises or is associated with a diagnostic label. Incertain exemplary embodiments, the diagnostic label is selected from thegroup consisting of: radiopharmaceutical or radioactive isotopes forgamma scintigraphy and PET, contrast agent for Magnetic ResonanceImaging (MRI), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves and fluorophores. Incertain exemplary embodiments, the conjugate is further monitored invivo.

In another aspect, the invention provides a method of administering aconjugate of the invention to an animal, comprising preparing an aqueousformulation of said conjugate and parenterally injecting saidformulation in the animal. In certain exemplary embodiments, theconjugate comprises a biologically active modifier. In certain exemplaryembodiments, the conjugate comprises a detectable modifier.

In another aspect, the invention provides a method of administering aconjugate of the invention to an animal, comprising preparating animplant comprising said conjugate, and implanting said implant into theanimal. In certain exemplary embodiments, the implant is a biodegradablegel matrix.

In another aspect, the invention provides a method for treating of ananimal in need thereof, comprising administering a conjugate accordingto the methods described above, wherein said conjugate is associatedwith a biologically active component.

In another aspect, the invention provides a method for treating of ananimal in need thereof, comprising administering a conjugate accordingto the methods described above, wherein said conjugate comprises abiologically active modifier. In certain exemplary embodiments, thebiologically active component is a gene vector.

In another aspect, the invention provides a method for eliciting animmune response in an animal, comprising administering a conjugate as inthe methods described above, wherein said conjugate comprises an antigenmodifier.

In another aspect, the invention provides a method of diagnosing adisease in an animal, comprising steps of:

-   -   administering a conjugate as in the methods described above,        wherein said conjugate comprises a detectable modifier; and    -   detecting the detectable modifier.

In certain exemplary embodiments, the step of detecting the detectablemodifier is performed non-invasively. In certain exemplary embodiments,the step of detecting the detectable modifier is performed usingsuitable imaging equipment.

DEFINITIONS

Certain compounds of the present invention, and definitions of specificfunctional groups are also described in more detail herein. For purposesof this invention, the chemical elements are identified in accordancewith the Periodic Table of the Elements, CAS version, Handbook ofChemistry and Physics, 75^(th) Ed., inside cover, and specificfunctional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in “OrganicChemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999,the entire contents of which are incorporated herein by reference.Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups. By the term “protecting group”, has used herein, itis meant that a particular functional moiety, e.g., O, S, or N, istemporarily blocked so that a reaction can be carried out selectively atanother reactive site in a multifunctional compound. In preferredembodiments, a protecting group reacts selectively in good yield to givea protected substrate that is stable to the projected reactions; theprotecting group must be selectively removed in good yield by readilyavailable, preferably nontoxic reagents that do not attack the otherfunctional groups; the protecting group forms an easily separablederivative (more preferably without the generation of new stereogeniccenters); and the protecting group has a minimum of additionalfunctionality to avoid further sites of reaction. As detailed herein,oxygen, sulfur, nitrogen and carbon protecting groups may be utilized.For example, in certain embodiments, certain exemplary oxygen protectinggroups may be utilized. These oxygen protecting groups include, but arenot limited to methyl ethers, substituted methyl ethers (e.g., MOM(methoxymethyl ether), MTM (methylthiomethyl ether), BOM(benzyloxymethyl ether), PMBM (p-methoxybenzyloxymethyl ether), to namea few), substituted ethyl ethers, substituted benzyl ethers, silylethers (e.g., TMS (trimethylsilyl ether), TES (triethylsilylether), TIPS(triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether, TBDPS (t-butyldiphenyl silyl ether), to name a few), esters(e.g., formate, acetate, benzoate (Bz), trifluoroacetate,dichloroacetate, to name a few), carbonates, cyclic acetals and ketals.In certain other exemplary embodiments, nitrogen protecting groups areutilized. These nitrogen protecting groups include, but are not limitedto, carbamates (including methyl, ethyl and substituted ethyl carbamates(e.g., Troc), to name a few) amides, cyclic imide derivatives, N-Alkyland N-Aryl amines, imine derivatives, and enamine derivatives, to name afew. Certain other exemplary protecting groups are detailed herein,however, it will be appreciated that the present invention is notintended to be limited to these protecting groups; rather, a variety ofadditional equivalent protecting groups can be readily identified usingthe above criteria and utilized in the present invention. Additionally,a variety of protecting groups are described in “Protective Groups inOrganic Synthesis” Third Ed. Greene, T. W. and Wuts, P. G., Eds., JohnWiley & Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe compounds that exert minimal destructive or host responseeffects while in contact with body fluids or living cells or tissues.Thus a biocompatible group, as used herein, refers to an aliphatic,alicyclic, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety,which falls within the definition of the term biocompatible, as definedabove and herein. The term “Biocompatibility” as used herein, is alsotaken to mean minimal interactions with recognition proteins, e.g.,naturally occurring antibodies, cell proteins, cells and othercomponents of biological systems. However, substances and functionalgroups specifically intended to cause the above effects, e.g., drugs andprodrugs, are considered to be biocompatible. Preferably, compounds are“biocompatible” if their addition to normal cells in vitro, atconcentrations similar to the intended in vivo concentration, results inless than or equal to 5% cell death during the time equivalent to thehalf-life of the compound in vivo (e.g., the period of time required for50% of the compound administered in vivo to be eliminated/cleared), andtheir administration in vivo induces minimal inflammation, foreign bodyreaction, immunotoxicity, chemical toxicity or other such adverseeffects. In the above sentence, the term “normal cells” refers to cellsthat are not intended to be destroyed by the compound being tested. Forexample, non-transformed cells should be used for testingbiocompatibility of antineoplastic compounds.

“Biodegradable”: As used herein, “biodegradable” polymers are polymersthat are susceptible to biological processing in vivo. As used herein,“biodegradable” compounds are those that, when taken up by cells, can bebroken down by the lysosomal or other chemical machinery or byhydrolysis into components that the cells can either reuse or dispose ofwithout significant toxic effect on the cells. The degradation fragmentspreferably induce no or little organ or cell overload or pathologicalprocesses caused by such overload or other adverse effects in vivo.Examples of biodegradation processes include enzymatic and non-enzymatichydrolysis, oxidation and reduction. Suitable conditions fornon-enzymatic hydrolysis, for example, include exposure of thebiodegradable polyal conjugates to water at a temperature and a pH oflysosomal intracellular compartment. Biodegradation of polyal conjugatesof the present invention can also be enhanced extracellularly, e.g. inlow pH regions of the animal body, e.g. an inflamed area, in the closevicinity of activated macrophages or other cells releasing degradationfacilitating factors. In certain preferred embodiments, the effectivesize of the polymer at pH˜7.5 does not detectably change over 1 to 7days, and remains within 50% of the original polymer size for at leastseveral weeks. At pH˜5, on the other hand, the polymer preferablydetectably degrades over 1 to 5 days, and is completely transformed intolow molecular weight fragments within a two-week to several-month timeframe. Polymer integrity in such tests can be measured, for example, bysize exclusion HPLC. Although faster degradation may be in some casespreferable, in general it may be more desirable that the polymerdegrades in cells with the rate that does not exceed the rate ofmetabolization or excretion of polymer fragments by the cells. Inpreferred embodiments, the polymers and polymer biodegradationbyproducts are biocompatible.

“Hydrophilic”: The term “hydrophilic” as it relates to substituents onthe polymer monomeric units does not essentially differ from the commonmeaning of this term in the art, and denotes organic moieties whichcontain ionizable, polar, or polarizable atoms, or which otherwise maybe solvated by water molecules. Thus a hydrophilic group, as usedherein, refers to an aliphatic, alicyclic, heteroaliphatic,heteroalicyclic, aryl or heteroaryl moiety, which falls within thedefinition of the term hydrophilic, as defined above. Examples ofparticular hydrophilic organic moieties which are suitable include,without limitation, aliphatic or heteroaliphatic groups comprising achain of atoms in a range of between about one and twelve atoms,hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester,thioester, aldehyde, nitryl, isonitryl, nitroso, hydroxylamine,mercaptoalkyl, heterocycle, carbamates, carboxylic acids and theirsalts, sulfonic acids and their salts, sulfonic acid esters, phosphoricacids and their salts, phosphate esters, polyglycol ethers, polyamines,polycarboxylates, polyesters and polythioesters. In preferredembodiments of the present invention, at least one of the polymermonomeric units include a carboxyl group (COOH), an aldehyde group(CHO), a methylol (CH₂OH) or a glycol (for example, CHOH—CH₂OH orCH—(CH₂OH)₂).

“Hydrophilic”: The term “hydrophilic” as it relates to the polymers ofthe invention generally does not differ from usage of this term in theart, and denotes polymers comprising hydrophilic functional groups asdefined above. In a preferred embodiment, hydrophilic polymer is awater-soluble polymer. Hydrophilicity of the polymer can be directlymeasured through determination of hydration energy, or determinedthrough investigation between two liquid phases, or by chromatography onsolid phases with known hydrophobicity, such as, for example, C4 or C18.

“Biomolecules”: The term “biomolecules”, as used herein, refers tomolecules (e.g., proteins, amino acids, peptides, polynucleotides,nucleotides, carbohydrates, sugars, lipids, nucleoproteins,glycoproteins, lipoproteins, steroids, etc.) which belong to classes ofchemical compounds, whether naturally-occurring or artificially created(e.g., by synthetic or recombinant methods), that are commonly found incells and tissues. Specific classes of biomolecules include, but are notlimited to, enzymes, receptors, neurotransmitters, hormones, cytokines,cell response modifiers such as growth factors and chemotactic factors,antibodies, vaccines, haptens, toxins, interferons, ribozymes,anti-sense agents, plasmids, DNA, and RNA.

“Physiological conditions”: The phrase “physiological conditions”, asused herein, relates to the range of chemical (e.g., pH, ionic strength)and biochemical (e.g., enzyme concentrations) conditions likely to beencountered in the extracellular fluids of living tissues. For mostnormal tissues, the physiological pH ranges from about 7.0 to 7.4.Circulating blood plasma and normal interstitial liquid representtypical examples of normal physiological conditions.

“Polysaccharide”, “carbohydrate” or “oligosaccharide”: The terms“polysaccharide”, “carbohydrate”, or “oligosaccharide” are known in theart and refer, generally, to substances having chemical formula(CH₂O)_(n), where generally n>2, and their derivatives. Carbohydratesare polyhydroxyaldehydes or polyhydroxyketones, or change to suchsubstances on simple chemical transformations, such as hydrolysis,oxydation or reduction. Typically, carbohydrates are present in the formof cyclic acetals or ketals (such as, glucose or fructose). Said cyclicunits (monosaccharides) may be connected to each other to form moleculeswith few (oligosaccharides) or several (polysaccharides) monosaccharideunits. Often, carbohydrates with well defined number, types andpositioning of monosaccharide units are called oligosaccharides, whereascarbohydrates consisting of mixtures of molecules of variable numbersand/or positioning of monosaccharide units are called polysaccharides.The terms “polysaccharide”, “carbohydrate”, and “oligosaccharide”, areused herein interchangeably. A polysaccharide may include natural sugars(e.g., glucose, fructose, galactose, mannose, arabinose, ribose, andxylose) and/or modified sugars (e.g., 2′-fluororibose, 2′-deoxyribose,and hexose).

“Small molecule”: As used herein, the term “small molecule” refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis) that have a relatively low molecular weight.Preferred small molecules are biologically active in that they produce alocal or systemic effect in animals, preferably mammals, more preferablyhumans. Typically, small molecules have a molecular weight of less thanabout 1500 g/mol. In certain preferred embodiments, the small moleculeis a drug. Preferably, though not necessarily, the drug is one that hasalready been deemed safe and effective for use by the appropriategovernmental agency or body. For example, drugs for human use listed bythe FDA under 21 C.F.R. §§330.5, 331 through 361, and 440 through 460;drugs for veterinary use listed by the FDA under 21 C.F.R. §§500 through589, incorporated herein by reference, are all considered suitable foruse with the present hydrophilic polymers.

Classes of small molecule drugs that can be used in the practice of thepresent invention include, but are not limited to, vitamins, anti-AIDSsubstances, anti-cancer substances, antibiotics, immunosuppressants,anti-viral substances, enzyme inhibitors, neurotoxins, opioids,hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants,muscle relaxants and anti-Parkinson substances, anti-spasmodics andmuscle contractants including channel blockers, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, vasodilating agents, inhibitors of DNA, RNA or proteinsynthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal andnon-steroidal anti-inflammatory agents, anti-angiogenic factors,anti-secretory factors, anticoagulants and/or antithrombotic agents,local anesthetics, ophthalmics, prostaglandins, anti-depressants,anti-psychotic substances, anti-emetics, imaging agents. Many largemolecules are also drugs.

A more complete, although not exhaustive, listing of classes andspecific drugs suitable for use in the present invention may be found in“Pharmaceutical Substances: Syntheses, Patents, Applications” by AxelKleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the“Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”,Edited by Susan Budavari et al., CRC Press, 1996, both of which areincorporated herein by reference.

“Pharmaceutically useful group or entity”: As used herein, the termPharmaceutically useful group or entity refers to a compound or fragmentthereof, or an organic moiety which, when associated with the polyalconjugates of the present invention, can exert some biological ordiagnostic function or activity when administered to a subject, orenhance the therapeutic, diagnostic or preventive properties of thepolyal conjugates in biomedical applications, or improve safety, alterbiodegradation or excretion, or is detectable. Examples of suitablepharmaceutically useful groups or entities includehydrophilicity/hydrophobicity modifiers, pharmacokinetic modifiers,biologically active modifiers, detectable modifiers.

“Modifier”: As used herein, the term modifier refers to an organic,inorganic or bioorganic moiety that is covalently incorporated into acarrier. Modifiers can be small molecules or macromolecules, and canbelong to any chemical or pharmaceutical class, e.g., nucleotides,chemotherapeutic agents, antibacterial agents, antiviral agents,immunomodulators, hormones or analogs thereof, enzymes, inhibitors,alkaloids and therapeutic radionuclides. In certain embodiments,chemotherapeutic agents include, but are not limited to, topoisomerase Iand II inhibitors, alkylating agents, anthracyclines, doxorubicin,cisplastin, carboplatin, vincristine, mitromycine, taxol, camptothecin,antisense oligonucleotides, ribozymes, and dactinomycines. In certainembodiments, modifiers according to the invention include, but are notlimited to, biomolecules, small molecules, therapeutic agents,pharmaceutically useful groups or entities, macromolecules, diagnosticlabels, chelating agents, hydrophilic moieties, dispersants, chargemodifying agents, viscosity modifying agents, surfactants, coagulationagents and flocculants, to name a few. A modifier can have one or morepharmaceutical functions, e.g., biological activity and pharmacokineticsmodification. Pharmacokinetics modifiers can include, for example,antibodies, antigens, receptor ligands, hydrophilic, hydrophobic orcharged groups. Biologically active modifiers include, for example,therapeutic drugs and prodrugs, antigens, immunomodulators. Detectablemodifiers include diagnostic labels, such as radioactive, fluorescent,paramagnetic, superparamagnetic, ferromagnetic, X-ray modulating,X-ray-opaque, ultrosound-reflective, and other substances detectable byone of available clinical or laboratory methods, e.g., scintigraphy, NMRspectroscopy, MRI, X-ray tomography, sonotomography, photoimaging,radioimmunoassay. Viral and non-viral gene vectors are considered to bemodifiers.

“Macromolecule”: As used herein, the term macromolecule refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis) that have a relatively high molecular weight,generally above 1500 g/mole Preferred macromolecules are biologicallyactive in that they exert a biological function in animals, preferablymammals, more preferably humans. Examples of macromolecules includeproteins, enzymes, growth factors, cytokines, peptides, polypeptides,polylysine, proteins, lipids, polyelectrolytes, immunoglobulins, DNA,RNA, ribozymes, plasmids, and lectins. For the purpose of thisinvention, supramolecular constructs such as viruses and proteinassociates (e.g., dimers) are considered to be macromolecules. Whenassociated with the polyal conjugates of the invention, a macromoleculemay be chemically modified prior to being associated with saidbiodegradable biocompatible polyal conjugate.

“Diagnostic label”: As used herein, the term diagnostic label refers toan atom, group of atoms, moiety or functional group, a nanocrystal, orother discrete element of a composition of matter, that can be detectedin vivo or ex vivo using analytical methods known in the art. Whenassociated with a biodegradable biocompatible polyal conjugate of thepresent invention, such diagnostic labels permit the monitoring of thebiodegradable biocompatible polyal conjugate in vivo. On the other hand,constructs and compositions that include diagnostic labels can be usedto monitor biological functions or structures. Examples of diagnosticlabels include, without limitations, labels that can be used in medicaldiagnostic procedures, such as, radiopharmaceutical or radioactiveisotopes for gamma scintigraphy and Positron Emission Tomography (PET),contrast agent for Magnetic Resonance Imaging (MRI) (for exampleparamagnetic atoms and superparamagnetic nanocrystals), contrast agentfor computed tomography, contrast agent for X-ray imaging method, agentfor ultrasound diagnostic method, agent for neutron activation, andmoiety which can reflect, scatter or affect X-rays, ultrasounds,radiowaves and microwaves, fluorophores in various optical procedures,etc.

“Effective amount of a glycol-specific oxidizing agent”: as it relatesto the oxidative cleavage of the polysaccharides referred to in thepresent invention, the phrase effective amount of a glycol-specificoxidizing agent means an amount of the glycol-specific oxidizing agentthat provides oxidative opening of essentially all carbohydrate rings ofa polysaccharide.

“Protected hydrophilic group” and “Protected organic moiety” as theseterms are used herein, mean a chemical group which will not interferewith a chemical reaction that the carrier or carrier conjugate issubjected to. Examples of protected hydrophilic groups includecarboxylic esters, alkoxy groups, thioesters, thioethers, vinyl groups,haloalkyl groups, Fmoc-alcohols, etc.

“Aliphatic”: In general, the term aliphatic, as used herein, includesboth saturated and unsaturated, straight chain (i.e., unbranched) orbranched aliphatic hydrocarbons, which are optionally substituted withone or more functional groups, as defined below. As will be appreciatedby one of ordinary skill in the art, “aliphatic” is intended herein toinclude, but is not limited to, alkyl, alkenyl, alkynyl moieties. Thus,as used herein, the term “alkyl” includes straight and branched alkylgroups. An analogous convention applies to other generic terms such as“alkenyl”, “alkynyl” and the like. Furthermore, as used herein, theterms “alkyl”, “alkenyl”, “alkynyl” and the like encompass bothsubstituted and unsubstituted groups. In certain embodiments, as usedherein, “lower alkyl” is used to indicate those alkyl groups(substituted, unsubstituted, branched or unbranched) having 1-6 carbonatoms. In certain embodiments, the alkyl, alkenyl and alkynyl groupsemployed in the invention contain 1-20 aliphatic carbon atoms. Incertain other embodiments, the alkyl, alkenyl, and alkynyl groupsemployed in the invention contain 1-10 aliphatic carbon atoms. In yetother embodiments, the alkyl, alkenyl, and alkynyl groups employed inthe invention contain 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-6 aliphatic carbon atoms. In yet other embodiments,the alkyl, alkenyl, and alkynyl groups employed in the invention contain1-4 carbon atoms.

Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl,isobutyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl,n-hexyl, sec-hexyl, moieties and the like, which again, may bear one ormore substituents, as previously defined. Alkenyl groups include, butare not limited to, for example, ethenyl, propenyl, butenyl,1-methyl-2-buten-1-yl, and the like. Representative alkynyl groupsinclude, but are not limited to, ethynyl, 2-propynyl (propargyl),1-propynyl and the like.

“Alicyclic”: The term alicyclic, as used herein, refers to compoundswhich combine the properties of aliphatic and cyclic compounds andinclude but are not limited to cyclic, or polycyclic aliphatichydrocarbons and bridged cycloalkyl compounds, which are optionallysubstituted with one or more functional groups, as defined below. Aswill be appreciated by one of ordinary skill in the art, “alicyclic” isintended herein to include, but is not limited to, cycloalkyl,cycloalkenyl, and cycloalkynyl moieties, which are optionallysubstituted with one or more functional groups. Illustrative alicyclicgroups thus include, but are not limited to, for example, cyclopropyl,—CH₂-cyclopropyl, cyclobutyl, —CH₂-cyclobutyl, cyclopentyl,—CH₂-cyclopentyl-n, cyclohexyl, —CH₂-cyclohexyl, cyclohexenylethyl,cyclohexanylethyl, norborbyl moieties and the like, which again, maybear one or more substituents.

“Heteroaliphatic”: The term “heteroaliphatic”, as used herein, refers toaliphatic moieties in which one or more carbon atoms in the main chainhave been substituted with an heteroatom. Thus, a heteroaliphatic grouprefers to an aliphatic chain which contains one or more oxygen sulfur,nitrogen, phosphorus or silicon atoms, e.g., in place of carbon atoms.Heteroaliphatic moieties may be saturated or unsaturated, branched orlinear (i.e., unbranched), and substituted or unsubstituted.Substituents include, but are not limited to, any of the substitutentsmentioned below, i.e., the substituents recited below resulting in theformation of a stable compound.

“Heteroalicyclic”: The term heteroalicyclic, as used herein, refers tocompounds which combine the properties of heteroaliphatic and cycliccompounds and include but are not limited to saturated and unsaturatedmono- or polycyclic heterocycles such as morpholino, pyrrolidinyl,furanyl, thiofuranyl, pyrrolyl etc., which are optionally substitutedwith one or more functional groups. Substituents include, but are notlimited to, any of the substitutents mentioned below, i.e., thesubstituents recited below resulting in the formation of a stablecompound.

“Alkyl”: the term alkyl as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom, which alkyl groups are optionally substituted with one ormore functional groups. Substituents include, but are not limited to,any of the substitutents mentioned below, i.e., the substituents recitedbelow resulting in the formation of a stable compound. Examples of alkylradicals include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, n-heptyl,n-octyl, n-decyl, n-undecyl, and dodecyl.

“Alkoxy”: the term alkoxy as used herein refers to an alkyl groups, aspreviously defined, attached to the parent molecular moiety through anoxygen atom. Examples include, but are not limited to, methoxy, ethoxy,propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy.

“Alkenyl”: the term alkenyl denotes a monovalent group derived from ahydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen atom, which alkenyl groups are optionallysubstituted with one or more functional groups. Substituents include,but are not limited to, any of the substitutents mentioned below, i.e.,the substituents recited below resulting in the formation of a stablecompound. Alkenyl groups include, for example, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, and the like.

“Alkynyl”: the term alkynyl as used herein refers to a monovalent groupderived form a hydrocarbon having at least one carbon-carbon triple bondby the removal of a single hydrogen atom, which alkenyl groups areoptionally substituted. Substituents include, but are not limited to,any of the substitutents mentioned below, i.e., the substituents recitedbelow resulting in the formation of a stable compound. Representativealkynyl groups include ethynyl, 2-propynyl (propargyl), 1-propynyl, andthe like.

“Amine”: the term amine as used herein refers to one, two, or three,respectively, alkyl groups, as previously defined, attached to theparent molecular moiety through a nitrogen atom. The term alkylaminorefers to a group having the structure —NHR′ wherein R′ is an alkylgroup, as previously defined; and the term dialkylamino refers to agroup having the structure —NR′R″, wherein R′ and R″ are eachindependently selected from the group consisting of alkyl groups. Theterm trialkylamino refers to a group having the structure —NR′R″R′″,wherein R′, R″, and R′″ are each independently selected from the groupconsisting of alkyl groups. Additionally, R′, R″, and/or R′″ takentogether may optionally be —(CH₂)_(k)— where k is an integer from 2 to6. Example include, but are not limited to, methylamino, dimethylamino,ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino,iso-propylamino, piperidino, trimethylamino, and propylamino.

“Aryl”: The term aryl, as used herein, refers to stable mono- orpolycyclic, unsaturated moieties having preferably 3-14 carbon atoms,each of which may be substituted or unsubstituted. Substituents include,but are not limited to, any of the substitutents mentioned below, i.e.,the substituents recited below resulting in the formation of a stablecompound. The term aryl may refer to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.

“Heteroaryl”: The term heteroaryl, as used herein, refers to a stableheterocyclic or polyheterocyclic, unsaturated radical having from fiveto ten ring atoms of which one ring atom is selected from S, O and N;zero, one or two ring atoms are additional heteroatoms independentlyselected from S, O and N; and the remaining ring atoms are carbon, theradical being joined to the rest of the molecule via any of the ringatoms. Heteroaryl moieties may be substituted or unsubstituted.Substituents include, but are not limited to, any of the substitutentsmentioned below, i.e., the substituents recited below resulting in theformation of a stable compound. Examples of heteroaryl nuclei includepyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl,thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl,furanyl, quinolinyl, isoquinolinyl, and the like.

It will also be appreciated that aryl and heteroaryl moieties, asdefined herein may be attached via an aliphatic, alicyclic,heteroaliphatic, heteroalicyclic, alkyl or heteroalkyl moiety and thusalso include -aliphatic)aryl, -(heteroaliphatic)aryl,-(aliphatic)heteroaryl, -(heteroaliphatic)heteroaryl, -(alkyl)aryl,-(heteroalkyl)aryl, -heteroalkyl)aryl, and -(heteroalkyl)heteroarylmoieties. Thus, as used herein, the phrases “aryl or heteroaryl” and“aryl, heteroaryl, -(aliphatic)aryl, -(heteroaliphatic)aryl,-(aliphatic)heteroaryl, (heteroaliphatic)heteroaryl, -alkyl)aryl,-(heteroalkyl)aryl, -(heteroalkyl)aryl, and -(heteroalkyl)heteroaryl”are interchangeable.

“Carboxylic acid”: The term carboxylic acid as used herein refers to agroup of formula —CO₂H.

“Halo, halide and halogen”: The terms halo, halide and halogen as usedherein refer to an atom selected from fluorine, chlorine, bromine, andiodine.

“Methylol”: The term methylol as used herein refers to an alcohol groupof the structure CH₂OH.

“Hydroxyalkyl”: As used herein, the term hydroxyalkyl refers to an alkylgroup, as defined above, bearing at least one OH group.

“Mercaptoalkyl”: The term mercaptoalkyl as used therein refers to analkyl group, as defined above, bearing at least one SH group

“Heterocyclic”: The term heterocyclic, as used herein, refers to anon-aromatic partially unsaturated or fully saturated 3- to 10-memberedring system, which includes single rings of 3 to 8 atoms in size and bi-and tri-cyclic ring systems which may include aromatic six-membered arylor aromatic heterocyclic groups fused to a non-aromatic ring.Heterocyclic moieties may be substituted or unsubstituted. Substituentsinclude, but are not limited to, any of the substitutents mentionedbelow, i.e., the substituents recited below resulting in the formationof a stable compound. Heterocyclic rings include those having from oneto three heteroatoms independently selected from oxygen, sulfur, andnitrogen, in which the nitrogen and sulfur heteroatoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized.

“Acyl”: The term acyl, as used herein, refers to a group comprising acarbonyl group of the formula C═O. Examples of acyl groups includealdehydes, ketones, carboxylic acids, acyl halides, anhydrides,thioesters, amides and carboxylic esters.

“Hydrocarbon”: The term hydrocarbon, as used herein, refers to anychemical group comprising hydrogen and carbon. The hydrocarbon may besubstituted or unsubstitued. The hydrocarbon may be unsaturated,saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.Illustrative hydrocarbons include, for example, methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl,cyclohexyl, methoxy, diethylamino, and the like. As would be known toone skilled in this art, all valencies must be satisfied in making anysubstitutions.

“Substituted”: The terms substituted, whether preceded by the term“optionally” or not, and substituent, as used herein, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. When more than one position in any givenstructure may be substituted with more than one substituent selectedfrom a specified group, the substituent may be either the same ordifferent at every position. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. Heteroatoms such as nitrogen may have hydrogen substituentsand/or any permissible substituents of organic compounds describedherein which satisfy the valencies of the heteroatoms. Examples ofsubstituents include, but are not limited to aliphatic; alicyclic;heteroaliphatic; heteroalicyclic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R)₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x) whereineach occurrence of R_(x) independently includes, but is not limited to,aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl,heteroaryl, alkylaryl, or alkylheteroaryl, wherein any of the aliphatic,alicyclic, heteroaliphatic, heteroalicyclic, alkylaryl, oralkylheteroaryl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted.

The following are more general terms used throughout the presentapplication:

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, at any stage of development, including, for example,mammals, birds, reptiles, amphibians, fish, worms and single cells. Cellcultures and live tissue samples are considered to be pluralities ofanimals. Preferably, the non-human animal is a mammal (e.g., a rodent, amouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). Ananimal may be a transgenic animal or a human clone.

“Associated with”: When two entities are “associated with” one anotheras described herein, they are linked by a direct or indirect covalent ornon-covalent interaction. Preferably, the association is covalent.Desirable non-covalent interactions include hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions,electrostatic interactions, or combinations thereof, etc.

“Effective amount”: In general, as it refers to an active agent or drugdelivery device, the term “effective amount” refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof an agent or device may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the composition of theencapsulating matrix, the target tissue, etc. For example, the effectiveamount of microparticles containing an antigen to be delivered toimmunize an individual is the amount that results in an immune responsesufficient to prevent infection with an organism having the administeredantigen.

“PHF” refers to poly(1-hydroxymethylethylene hydroxymethyl-formal).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the blood activity following iv injection of¹¹¹In-labeled trypsin conjugates. (Δ) DTPA-modified trypsin; (□)PHF-SA-Trypsin; and (♦) PHF-AO-Trypsin conjugates.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

Certain preferred embodiments of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Principlefeatures of the invention may be employed in various embodiments withoutdeparting from the scope of the invention.

In one aspect, the present invention provides conjugates of small andlarge (bio)molecules and/or other (in)organic moieties (i.e., modifiers)with carriers, wherein the small/larger (bio)molecules and/or other(in)organic moieties are covalently attached to the carrier viaoxime-containing linkages. In certain embodiments, the carrier is amacromolecule, a molecular matrix or an interface. In certain otherembodiments, the carrier is a fully synthetic, semi-synthetic ornaturally-occurring polymer. In certain exemplary embodiments; theconjugates of the invention find use in biomedical applications, such asgene and drug delivery and tissue engineering, and the carrier isbiocompatible and biodegradable. In certain other embodiments, thecarrier is hydrophilic. In certain exemplary embodiments, the polymercarriers used in the present invention comprise at least onehydrolizable bond in each monomer unit positioned within the main chain.This ensures that the degradation process (via hydrolysis/cleavage ofthe monomer units) will result in fragmentation of the polymer conjugateto the monomeric components (i.e., degradation), and confers to thepolymer conjugates of the invention their biodegradable properties. Theproperties (e.g., solubility, bioadhesivity and hydrophilicity) ofbiodegradable biocompatible polymer conjugates can be modified bysubsequent substitution of additional hydrophilic or hydrophobic groups.

Non-bioadhesive, fully biodegradable soluble polymer conjugates would beinstrumental in such biomedical applications. However, rationaldevelopment of such materials is hindered by the complexity ofmacromolecule interactions with the biological milieu. Addressing theneed for polymer conjugates having these above characteristics, thepresent invention provides novel biodegradable biocompatible polyalconjugates, which are chemically modified by covalent attachment ofsmall/large (bio)molecules or other (in)organic moieties (i.e.,modifiers) via oxime-containing linkages.

Biodegradable Biocompatible Polyal Conjugates

As discussed above, novel concepts in pharmacology and bioengineeringimpose new, more specific and more stringent requirements on biomedicalpolymers. Ideally, advanced macromolecular materials would combinenegligible reactivity in vivo with low toxicity and biodegradability.Polymer structure should support an ample set of technologies forpolymer derivatization, for example, conjugation with drugs,cell-specific ligands, or other desirable modifiers. Materials combiningall the above features would be useful in the development ofmacromolecular drugs, drug delivery systems, implants and templates fortissue engineering.

On the chemistry level, developing such biocompatible and biodegradablematerials translates into developing macromolecules with minimizedinteractions in vivo, main chains susceptible to hydrolysis (e.g.,degradation) in vivo, and readily modifiable functional groups. Anotherconsideration to take into account is that both the main chain and thefunctional groups interact with an extremely complex biological milieu,and all interactions may be amplified via cooperative mechanisms.

Biomolecule interactions in vivo are mediated by several components ofcell surfaces, extracellular matrix, and biological fluids. For example,both biomolecule internalization by cells and cell adhesion topolymer-coated surfaces can be mediated by several cell surfaceelements, many of which are functionally specialized. Cooperativebinding, often referred to as “non-specific interactions”, is anothermajor factor of biomolecule (and surface) reactivity in vivo. Cellinteractions with polymers and recognition protein-polymer complexesalso have an element of cooperativity. The very nature of cooperativeinteractions in complex systems suggests that any large molecule cansignificantly interact with a complex substrate for the simple reasonthat, because the binding energy is additive, the association constantof cooperative binding (K_(a)) would grow with the number ofassociations exponentially. In other words, any polymer of a sufficientlength can be expected to interact with at least one of the variouscomponents of a biological system. Even if a molecule of certain sizeshows low interactions in cell cultures and in vivo, a larger moleculeof the same type (or a supra molecular assembly) can have a much higherbinding activity.

In summary, even if polymer molecules are assembled of domains that donot interact with cell receptors and recognition proteins, suchmolecules can be capable of cooperative interactions in vivo; i.e.,completely inert polymers may not exist at all. However, severalbiomolecules and biological interfaces do appear to be functionallyinert, except for their specialized signaling domains. For example,plasma proteins are known to circulate for several weeks without uptakein the reticuloendothelial system (RES), unlike artificial constructs ofcomparable size that have never been reported to have comparable bloodhalf-lives. Without wishing to be bound to any particular theory, wepropose that the mutual “inertness” of natural biomolecules and surfacesmay relate to their relatively uniform interface structures, where thepotential binding sites are always saturated by naturally occurringcounteragents present in abundance. Therefore, emulation of the commoninterface structures can result in a material that would not activelyinteract with actually existing binding sites because these sites wouldbe pre-occupied by the natural “prototypes”.

Poly- and oligosaccharides are the most abundant interface moleculesexpressed (as varous glycoconjugates) on cell surfaces, plasma proteins,and proteins of the extracellular matrix. Therefore, the inventionencompasses structural emulation of interface carbohydrates in an effortto identify and exclude all structural components that can berecognized, even with low affinity, by any biomolecule, especially bycell receptors and recognition proteins.

All interface carbohydrates have common structural domains which appearto be irrelevant to their biological function. The acetal/ketal groupand the adjacent atoms are present in all carbohydrates regardless ofbiological activity, whereas the receptor specificity of each moleculedepends on the structure and configuration of the glycol domains of thecarbohydrate rings. Thus it would seem that biologically inert(“stealth”) polymers could be obtained using substructures that form theacetal/ketal structures of the carbohydrate rings; i.e., the O—C—O—group and adjacent carbons. Although functional groups that are commonin naturally occurring glycoconjugates (e.g., OH groups) can be used assubstitutents, the potentially biorecognizable combinations of thesegroups, such as rigid structures at C1-C2-C3-C4 (in pyranoses) is notdesirable.

The present invention is founded on the recognition that themacromolecular products of the cleavage of at least one of thecarbon-carbon bonds in the C1-C2-C3-C4 portion in substantially all thecarbohydrate rings of a polysaccharide would have the desired properties(e.g., an essentially inert biocompatible hydrophilic polymer). Inaddition, synthetic strategies designed to position the polysaccharideacetal/ketal groups within the main chain of the resultingmacromolecular product would ensure degradability via proton-catalyzedhydrolysis.

Biocompatible biodegradable polyacetals and polyketals according to thisconcept have been described in U.S. Pat. Nos. 5,811,510; 5,863,990 and5,958,398; U.S. Provisional Patent Application 60/348,333; EuropeanPatent No.: 0820473; and International Patent ApplicationPCT/US03/01017, each of the above listed patent documents isincorporated herein by reference in its entirety.

The present invention encompasses biodegradable biocompatiblehydrophilic polyal conjugates, as well as methods of preparation andmethods of use thereof. In certain embodiments, it is anticipated thatthe present invention will be particularly useful in combination withthe above-referenced patent documents, as well as U.S. Pat. No.5,582,172; U.S. Patent Application No.: 60/147,919 and Ser. No.09/634,320, each of the above listed patent documents is incorporatedherein by reference in its entirety.

As described in Examples 3 and 4, we have successfully madebiodegradable biocompatible polyal conjugates which are hydrophilic,hydrolyzable and comprise modifiers (e.g., pharmaceutically usefulgroups) covalently attached to the polymer carrier via oxime-containinglinkages. In certain exemplary embodiments, the polyal conjugates of thepresent invention have at least one acetal/ketal oxygen atom in eachmonomer unit positioned within the main chain. This ensures that thedegradation process (via hydrolysis/cleavage of the polymer acetal/ketalgroups) will result in fragmentation of the polyal conjugate to themonomeric components (i.e., degradation), and confers to the polyalconjugates of the invention their biodegradable properties. Theproperties (e.g., solubility, bioadhesivity and hydrophilicity) ofbiodegradable biocompatible polyal conjugates can be adjusted byincorporation of suitable hydrophilic or hydrophobic modifiers or bysubsequent substitution of additional hydrophilic or hydrophobic groups.The novelty of the present invention relates in part to the structureand properties of polyal conjugates comprising one or more modifierscovalently attached via oxime-containing linkages to a hydrophilicpolykal carrier having acetal/ketal groups in the main chain.

Thus, in certain embodiments, the invention provides a conjugatecomprising a carrier substituted with one or more occurrences of amoiety having the structure:

-   -   wherein each occurrence of M is independently a modifier; and    -   each occurrence of L^(M) is independently an oxime-containing        linker.

In certain embodiments, each occurrence of L^(M) is independently amoiety having the structure:

-   -   wherein each occurrence of L^(M1) is independently a substituted        or unsubstituted, cyclic or acyclic, linear or branched        C₀₋₁₂alkylidene or C₀₋₁₂alkenylidene moiety wherein up to two        non-adjacent methylene units are independently optionally        replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),        NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,        NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1),        NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein each occurrence of        R^(Z1) and R^(Z2) is independently hydrogen, alkyl, heteroalkyl,        aryl, heteroaryl or acyl.

In certain exemplary embodiments, one or more occurrences of L^(M)independently comprise a crosslinker adapted to facilitate attachment ofthe modifier M and/or the carrier onto L^(M). In certain embodiments,L^(M) comprises a functional group feasible for selective conjugationwith a chemical moiety present either in the carrier or in the modifier.For example, L^(M) may comprise an active ester (e.g.,N-hydroxysuccinimide, tetrafluorophenyl, or nitrophenyl ester) usefulfor conjugation with aminogroups. As another example, L^(M) may comprisea maleimido group feasible for conjugation with thiols.

Crosslinkers suited for practicing this embodiment of invention arewidely known in the art (see, for example, 1994 Pierce TechnicalHandbook: cross-linking (Appendix A), also available atwww.piercenet.com/resources/browse.cfm? fldID=184), includingbromoacetic NHS ester, 6-(iodoacetamido)caproic acid NHS ester,maleimidoacetic acid NHS ester, maleimidobenzoic acid NHS ester, etc.

In certain other embodiments, one or more occurrences of L^(M1)independently comprises a maleimide- or N-hydroxysuccinimideester-containing crosslinker. In yet other embodiments, one or moreoccurrences of L^(M1) independently comprises a N-maleimidoalkylcarboxylate (1), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (2),m-maleimidobenzoyl (3), or a 4-(p-maleimidophenyl)butyrate (4)crosslinker.

In still other embodiments, one or more occurrences of L^(M1)independently comprises a carboxysuccinimide crosslinker (5).

Carriers

In certain embodiments, biodegradable biocompatible polymer carriers,used for preparation of polymer conjugates of the invention, arenaturally occurring polysaccharides, glycopolysaccharides, and syntheticpolymers of polyglycoside, polyacetal, polyamide, polyether, andpolyester origin and products of their oxidation, fictionalization,modification, cross-linking, and conjugation.

In certain other embodiments, the carrier is a hydrophilic biodegradablepolymer selected from the group consisting of carbohydrates,glycopolysaccharides, glycolipids, glycoconjugates, polyacetals,polyketals, and derivatives thereof.

In certain exemplary embodiments, the carrier is a naturally occurringlinear and branched biodegradable biocompatible homopolysaccharideselected from the group consisting of cellulose, amylose, dextran,levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen andlixenan.

In certain other exemplary embodiments, the carrier is a naturallyoccurring linear and branched biodegradable biocompatibleheteropolysaccharide selected from the group consisting of agarose,hyluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginicacid and heparin.

In yet other exemplary embodiments, the carrier is a hydrophilic polymerselected from the group consisting of polyacrylates, polyvinyl polymers,polyesters, polyorthoesters, polyamides, polypeptides, and derivativesthereof.

In certain embodiments, the carrier comprises polysaccharides activatedby selective oxidation of cyclic vicinal diols of 1,2-, 1,4-, 1,6-, and2,6-pyranosides, and 1,2-, 1,5-, 1,6-furanosides, or by oxidation oflateral 6-hydroxy and 5,6-diol containing polysaccharides prior toconjugation with one or more modifiers.

In one embodiment, the carriers of the invention comprise activatedhydrophilic biodegradable biocompatible polymer carriers comprising from0.1% to 100% of polyacetal moieties represented by the followingchemical structure:(—O—CH₂—CHR₁—O—CHR₂—)_(n)

-   -   wherein R₁ and R₂ are independently hydrogen, hydroxyl,        carbonyl, carbonyl-containing substituent, a biocompatible        organic moiety comprising one or more heteroatoms or a protected        hydrophilic functional group; and n is an integer from 1-5000.

In certain exemplary embodiments, the carriers of the present inventionare polyals, and comprise acetal/ketal groups within the main chain.Although it is not necessary that the entire acetal/ketal group bepositioned within the polymer backbone, it is desirable that at leastone of the acetal/ketal oxygen atoms belongs to the main chain.

Accordingly, in still other exemplary embodiments, the carrier comprisesa biodegradable biocompatible polyacetal wherein at least a subset ofthe polyacetal repeat structural units have the following chemicalstructure:

-   -   wherein for each occurrence of the n bracketed structure, one of        R¹ and R² is hydrogen, and the other is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation.

In further exemplary embodiments, the carrier comprises a biodegradablebiocompatible polyketal wherein at least a subset of the polyketalrepeat structural units have the following chemical structure:

-   -   wherein each occurrence of R¹ and R² is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation.

Examples of suitable organic moieties are aliphatic groups having achain of atoms in a range of between about one and twelve atoms,hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester,thioester, aldehyde, nitryl, isonitryl, nitroso, hydroxylamine,mercaptoalkyl, heterocycle, carbamates, carboxylic acids and theirsalts, sulfonic acids and their salts, sulfonic acid esters, phosphoricacids and their salts, phosphate esters, polyglycol ethers, polyamines,polycarboxylates, polyesters, polythioesters, pharmaceutically usefulgroups, a biologically active substance or a diagnostic label.

In certain embodiments, in the polyacetals and polyketals describeddirectly above, for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ includes comprises a functionalgroup that increases the polymer hydrophilicity or is adapted forcovalent binding to linker L^(M).

In certain embodiments, in the polyacetals and polyketals describeddirectly above, for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ includes comprises a carbonylgroup adapted for covalent binding to linker L^(M). In certain exemplaryembodiments, the polyacetals and polyketals described directly above,wherein at least one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonylgroup, are conjugated with one or more moieties having the structureH₂N—O-L¹; wherein each occurrence of L¹ comprises a modifier orcomprises a functional group adapted for covalent binding to a modifier.

In yet another embodiments, at least one of R¹, R², R³, R⁴, R⁵ and R⁶contains a chiral moiety.

In certain embodiments, the biodegradable biocompatible carriers of theinvention can be crosslinked. A suitable crosslinking agent has theformula X¹—(R)—X², where R is a spacer group and X¹ and X² are reactivegroups. X¹ and X² can be different or the same. The spacer group R maybe an aliphatic, alicyclic, heteroaliphatic, heteroalicyclic, aryl orheteroaryl moiety. Examples of suitable spacer groups includebiodegradable or nonbiodegradable groups, for example, aliphatic groups,carbon chains containing biodegradable inserts such as disulfides,esters, etc. The term “reactive group,” as it relates to X¹ and X²,means functional groups which can be connected by a reaction within thebiodegradable biocompatible polyals, thereby crosslinking thebiodegradable biocompatible polyals. Suitable reactive groups which formcrosslinked networks with the biodegradable biocompatible polyalsinclude epoxides, halides, tosylates, mesylates, carboxylates,aziridines, cyclopropanes, esters, N-oxysuccinimide esters, disulfides,anhydrides etc.

In certain exemplary embodiments, the carrier is a biodegradablebiocompatible polyketal that is crosslinked with epibromohydrin, orepichlorohydrin. In certain embodiments, the epibromohydrin orepichlorohydrin is present in an amount in the range of between aboutone and about twenty five percent by weight of the crosslinkedbiodegradable biocompatible polyketals.

Alternatively, the term “reactive” group as it relates to X¹ and X²means a nucleophilic group that can be reacted with an aldehydeintermediate of the biodegradable biocompatible polyals, therebycrosslinking the biodegradable biocompatible polykals. Suitable reactivegroups for the aldehyde intermediate include amines, thiols, polyols,alcohols, ketones, aldehydes, diazocompounds, boron derivatives, ylides,isonitriles, hydrazines and their derivatives and hydroxylamines andtheir derivatives, etc.

In one embodiment, the biodegradable biocompatible polyals of thepresent invention have a molecular weight of between about 0.5 and about1500 kDa. In a preferred embodiment of the present invention, thebiodegradable biocompatible polyals have a molecular weight of betweenabout 1 and about 1000 kDa.

In certain embodiments, the polymer carriers are modified (i.e.,conjugated with one or more modifiers) at one or both termini. Forexample, when the carrier is a polyketal, the carrier may have thestructure:

-   -   wherein n is an integer and R′, R″ and R′″ may be a modifier.        For example, R′ can comprise an N-hydroxysuccinimide ester or a        maleimide moiety for conjugation with proteins or other        biomolecules; R″ and R′″ can comprise a phospholipid and a        target specific moiety, such as antibody, respectively, for        liposome modification.

In certain other embodiments, carriers can be substituted at oneterminal and one or more non-terminal positions, or at both terminal andone or more non-terminal positions.

In certain embodiments, the carrier is a linear macromolecule, abranched macromolecule, a globular macromolecule, a graft copolymer, acomb copolymer, a nanoparticle or a lipid-based carrier. In certainexemplary embodiments, the lipid-based carrier is a liposome.

Modifiers

In certain embodiments, modifiers according to the invention include,but are not limited to, biomolecules, small molecules, therapeuticagents, microparticles, pharmaceutically useful groups or entities,macromolecules, diagnostic labels, chelating agents, hydrophilicmoieties, dispersants, charge modifying agents, viscosity modifyingagents, surfactants, coagulation agents and flocculants, to name a few.

Examples of biomolecules include, but are not limited to, enzymes,receptors, neurotransmitters, hormones, cytokines, cell responsemodifiers such as growth factors and chemotactic factors, antibodies,vaccines, haptens, toxins, interferons, ribozymes, anti-sense agents,plasmids, DNA, and RNA.

Examples of small molecules include, but are not limited to, drugs suchas vitamins, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics and imagingagents.

Examples of suitable pharmaceutically useful groups or entities include,but are not limited to, hydrophilicity/hydrophobicity modifiers,pharmacokinetic modifiers, biologically active modifiers and detectablemodifiers.

Examples of diagnostic labels include, but are not limited to,diagnostic radiopharmaceutical or radioactive isotopes for gammascintigraphy and PET, contrast agent for Magnetic Resonance Imaging(MRI) (for example paramagnetic atoms and superparamagneticnanocrystals), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, and moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves, fluorophores in variousoptical procedures, etc. Diagnostic radiopharmaceuticals includeγ-emitting radionuclides, e.g., indium-111, technetium-99m andiodine-131, etc. Contrast agents for MRI (Magnetic Resonance Imaging)include magnetic compounds, e.g. paramagnetic ions, iron, manganese,gadolinium, lanthanides, organic paramagnetic moieties andsuperparamagnetic, ferromagnetic and antiferromagnetic compounds, e.g.,iron oxide colloids, ferrite colloids, etc. Contrast agents for computedtomography and other X-ray based imaging methods include compoundsabsorbing X-rays, e.g., iodine, barium, etc. Contrast agents forultrasound based methods include compounds which can absorb, reflect andscatter ultrasound waves, e.g., emulsions, crystals, gas bubbles, etc.Still other examples include substances useful for neutron activation,such as boron and gadolinium. Further, labels can be employed which canreflect, refract, scatter, or otherwise affect X-rays, ultrasound,radiowaves, microwaves and other rays useful in diagnostic procedures.Fluorescent labels can be used for photoimaging. In certain embodimentsa modifier comprises a paramagnetic ion or group.

In certain embodiments, a modifier of the invention comprises at leastone functional group suitable for covalent binding with a carbonyl grouppresent on a carrier via an oxime linkage.

Alternatively, or additionally, a modifier may be adapted for covalentbinding with a carbonyl group present on a carrier via an oxime linkage.For example, a modifier may be covalently attached to a linker moietycomprising a functional group that can form an oxime linkage with acarbonyl group present on a carrier. For instance, the inventivemodifiers may have the structure:

-   -   wherein M is a modifier; L^(M1) is a substituted or        unsubstituted, cyclic or acyclic, linear or branched        C₀₋₁₂alkylidene or C₀₋₁₂alkenylidene moiety wherein up to two        non-adjacent methylene units are independently optionally        replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),        NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,        NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1),        NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein each occurrence of        R^(Z1) and R^(Z2) is independently hydrogen, alkyl, heteroalkyl,        aryl, heteroaryl or acyl; and R^(N1) and R^(N2) are        independently hydrogen, an aliphatic, alicyclic,        heteroaliphatic, heterocyclic, aryl or heteroaryl moiety, or a        nitrogen protecting group, or R^(N1) and R^(N2), taken together        with the nitrogen atom to which they are attached, form a        substituted or unsubstituted heterocyclic or heteroaryl moiety.

In certain exemplary embodiments, L^(M1) comprises an NHS estercrosslinker and the compound has the structure:

-   -   wherein p is 0-5.

In certain other exemplary embodiments, L¹ comprises a maleimidecrosslinker and the compound has the structure:

-   -   wherein p₁ and p₂ are independently integers from 1-5.

In certain exemplary embodiments, in the compounds having the structure:

R^(N1)R^(N2)N— is a moiety having the structure:

In other exemplary embodiments, R^(N1)R^(N2)N— is —NH₂.

In another aspect, the invention provides bifunctional compoundssuitable for covalently binding a carrier and one or more modifiers viaoxime linkages. In certain embodiments, the bifunctional compoundscomprise (i) a functional group suitable for covalent binding with acarbonyl group present on a carrier via an oxime linkage; and (ii) afunctional group Q suitable for covalent attachment to a modifier.

In certain exemplary embodiments, the inventive bifunctional compoundshave the structure R^(N1)R^(N2)N—O-L¹; wherein R^(N1) and R^(N2) areindependently hydrogen, an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aryl or heteroaryl moiety, or a nitrogen protecting group,or R^(N1) and R^(N2), taken together with the nitrogen atom to whichthey are attached, form a substituted or unsubstituted heterocyclic orheteroaryl moiety; and L¹ is an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aryl or heteroaryl moiety comprising a functional groupadapted for covalent binding to a modifier.

In certain exemplary embodiments, L¹ is a moiety having the structure—(CR^(L1)R^(L2))_(p)-Q-, wherein p is an integer from 0-6, R^(L1) andR^(L2) are independently hydrogen, an aliphatic, alicyclic,heteroaliphatic, heterocyclic, aryl or heteroaryl moiety or WR^(W1)wherein W is O, S, NH, CO, SO₂, COO, CONH, and R^(W1) is hydrogen, analiphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl,alkylaryl or alkylheteroaryl moiety, and Q is a moiety comprising afunctional group adapted for covalent binding to a modifier. In certainother exemplary embodiments, L¹ is —CH₂)_(p) wherein p is an integerfrom 0-5. In yet other exemplary embodiments, L¹ is —CH₂)_(p),—CH(OH)CH₂NH— wherein p₁ is an integer from 1-5.

In certain embodiments, Q comprises an active ester moiety useful forconjugation with amino groups. In certain exemplary embodiments, Qcomprises N-hydroxy succinimide ester, tetrafluorophenyl ester ornitrophenyl ester. In certain other exemplary embodiments, Q comprises asuccinimidyl ester moiety having the structure:

In certain embodiments, Q comprises a maleimido moiety useful forconjugation with thio groups. In certain other exemplary embodiments, Qcomprises a succinimidyl ester moiety having the structure:

In certain other exemplary embodiments, L¹ is —(CH₂)_(p) wherein p is aninteger from 0-5; and Q a maleimidyl moiety having the structure:

-   -   wherein p₂ is an integer from 1-5.

In yet other exemplary embodiments, L¹ is —CH₂)_(p), —CH(OH)CH₂NH—wherein p₁ is an integer from 1-5, and Q is a maleimidyl moiety havingthe structure:

-   -   wherein p₂ is an integer from 1-5.

In certain embodiments, one of R^(N1) and R^(N2) is a nitrogenprotecting group. Nitrogen protecting groups, as well as protection anddeprotection methods are known in the art. Guidance may be found in“Protective Groups in Organic Synthesis” Third Ed. Greene, T. W. andWuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entirecontents of which are hereby incorporated by reference. In certainexemplary embodiments, R^(N1) and R^(N2) are each hydrogen.

In certain exemplary embodiments, in the bifunctional compounds of theinvention, R^(N1)R^(N2)N— is a moiety having the structure:

Conjugates

Conjugates of the invention comprise one or more occurrences of M, whereM is a modifier, wherein the one or more occurrences of M may be thesame or different. In certain embodiments, one or more occurrences of Mare biocompatible moieties. In certain embodiments, one or moreoccurrences of M are hydrophilic moieties.

In certain other embodiment, one or more occurrences of M comprise, orare attached to the carrier through, a biodegradable bond. In certainexemplary embodiments, the biodegradable bond is selected from the groupconsisting of acetal, ketal, amide, ester, thioester, enamine, inline,imide, dithio, and phosphoester bond.

In other embodiments, in the conjugates of the invention, one or moreoccurrences of M comprise a biologically active modifier. In certainexemplary embodiments, one or more occurrence of M is selected from thegroup consisting of proteins, antibodies, antibody fragments, peptides,antineoplastic drugs, hormones, cytokines, enzymes, enzyme substrates,receptor ligands, lipids, nucleotides, nucleosides, metal complexes,cations, anions, amines, heterocycles, heterocyclic amines, aromaticgroups, aliphatic groups, intercalators, antibiotics, antigens,immunomodulators, and antiviral compounds.

In certain other embodiments, one or more occurrence of M comprises adetectable label. In certain exemplary embodiments, one or moreoccurrence of M comprises atoms or groups of atoms comprisingradioactive, paramagnetic, superparamagnetic, fluorescent, or lightabsorbing structural domains.

In certain other embodiments, one or more occurrences of M comprise adiagnostic label. In certain exemplary embodiments, one or moreoccurrence of M comprises radiopharmaceutical or radioactive isotopesfor gamma scintigraphy and PET, contrast agent for Magnetic ResonanceImaging (MRI), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves, microwaves and/or fluorophores.

In certain exemplary embodiments, the inventive conjugate comprises abiologically active modifier and a detectable label.

The biodegradable biocompatible polykal conjugates of the invention canbe prepared to meet desired requirements of biodegradability andhydrophilicity. For example, under physiological conditions, a balancebetween biodegradability and stability can be reached. For instance, itis known that macromolecules with molecular weights beyond a certainthreshold (generally, above 50-100 kDa, depending on the physical shapeof the molecule) are not excreted through kidneys, as small moleculesare, and can be cleared from the body only through uptake by cells anddegradation in intracellular compartments, most notably lysosomes. Thisobservation exemplifies how functionally stable yet biodegradablematerials may be designed by modulating their stability under generalphysiological conditions (pH=7.5±0.5) and at lysosomal pH (pH near 5).For example, hydrolysis of acetal and ketal groups is known to becatalyzed by acids, therefore polyals will be in general less stable inacidic lysosomal environment than, for example, in blood plasma. One candesign a test to compare polymer degradation profile at, for example,pH=5 and pH=7.5 at 37° C. in aqueous media, and thus to determine theexpected balance of polymer stability in normal physiologicalenvironment and in the “digestive” lysosomal compartment after uptake bycells. Polymer integrity in such tests can be measured, for example, bysize exclusion HPLC. In many cases, it will be preferable that at pH=7.5the effective size of the polymer will not detectably change over 1 to 7days, and remain within 50% from the original for at least severalweeks. At pH=5, on the other hand, the polymer should preferablydetectably degrade over 1 to 5 days, and be completely transformed intolow molecular weight fragments within a two-week to several-month timeframe. Although faster degradation may be in some cases preferable, ingeneral it may be more desirable that the polymer degrades in cells withthe rate that does not exceed the rate of metabolization or excretion ofpolymer fragments by the cells.

Accordingly, the conjugates of the present invention are expected to bebiodegradable, in particular upon uptake by cells, and relatively“inert” in relation to biological systems. The products of degradationare preferably uncharged and do not significantly shift the pH of theenvironment. It is proposed that the abundance of alcohol groups mayprovide low rate of polymer recognition by cell receptors, particularlyof phagocytes. The polymer backbones of the present invention generallycontain few, if any, antigenic determinants (characteristic, forexample, for polysaccharides and polypeptides) and generally do notcomprise rigid structures capable of engaging in “key-and-lock” typeinteractions. Thus, the soluble, crosslinked and solid conjugates ofthis invention are predicted to have low toxicity and bioadhesivity,which makes them suitable for several biomedical applications.

In certain embodiments of the present invention, the biodegradablebiocompatible polyal conjugates can form linear or branched structures.The biodegradable biocompatible polyal conjugates of the presentinvention can be chiral (optically active). Optionally, thebiodegradable biocompatible polyal conjugates of the present inventioncan be racemic.

In yet another embodiment, the conjugates of the present invention areassociated with a macromolecule. Examples of suitable macromoleculesinclude, but are not limited to, enzymes, polypeptides, polylysine,proteins, lipids, polyelectrolytes, antibodies, ribonucleic anddeoxyribonucleic acids and lectins. The macromolecule may be chemicallymodified prior to being associated with said biodegradable biocompatibleconjugate. Circular and linear DNA and RNA (e.g., plasmids) andsupramolecular associates thereof, such as viral particles, for thepurpose of this invention are considered to be macromolecules. Incertain embodiments, conjugates of the invention are non-covalentlyassociated with macromolecules.

In certain embodiments, the conjugates of the invention arewater-soluble. In certain embodiments, the conjugates of the inventionare water-insoluble. In certain embodiments, the inventive conjugate isin a solid form. In certain embodiments, the conjugates of the inventionare colloids. In certain embodiments, the conjugates of the inventionare in particle form. In certain embodiments, the conjugates of theinvention are in gel form. In certain embodiments, the conjugates of theinvention are in a fiber form. In certain embodiments, the conjugates ofthe invention are in a film form.

Synthetic Methods

According to the present invention, any available techniques can be usedto make the inventive conjugates or compositions including them, andintermediates and components (e.g., carriers and modifiers) useful formaking them. For example, semi-synthetic and fully synthetic methodssuch as those discussed in detail below may be used.

Carriers

Methods for preparing polymer carriers (e.g., biocompatible,biodegradable polymer carriers) suitable for conjugation to modifiersare known in the art. For example, synthetic guidance can be found inU.S. Pat. Nos. 5,811,510; 5,863,990 and 5,958,398; U.S. ProvisionalPatent Application 60/348,333; European Patent No.: 0820473; andInternational Patent Application PCT/US03/01017. The skilledpractitioner will know how to adapt these methods to make polymercarriers for use in the practice of the invention.

Semi Synthetic Route

For example, semi-synthetic polyals may be prepared from polyaldoses andpolyketoses via complete lateral cleavage of carbohydrate rings withperiodate in aqueous solutions, with subsequent conjugation of aldehydegroups with one or more modifiers or conversion into hydrophilicmoieties, e.g. via borohydride reduction. In an exemplary embodiment,the carbohydrate rings of a suitable polysaccharide can be oxidized byglycol-specific reagents, resulting in the cleavage of carbon-carbonbonds between carbon atoms that are each connected to a hydroxyl group.An example of application of this methodology to dextran B-512 isillustrated below:

A similar approach may be used with Levan:

In one embodiment, a method for forming the biodegradable biocompatiblepolyal conjugates of the present invention comprises a process by whicha suitable polysaccharide is combined with an effective amount of aglycol-specific oxidizing agent to form an aldehyde intermediate. Thealdehyde intermediate, which is a polyal itself, may then be reactedwith one or more suitable modifiers to form a biodegradablebiocompatible polyal conjugate comprising oxime-containing linkages.

In certain exemplary embodiments, the carrier is a biodegradablebiocompatible polyacetal wherein at least a subset of the polyacetalrepeat structural units have the following chemical structure:

-   -   wherein for each occurrence of the n bracketed structure, one of        R¹ and R² is hydrogen, and the other is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R¹ and R⁶ comprises a carbonyl group suitable        for oxime formation. In certain embodiments, the carbonyl group        is an aldehyde moiety.

In yet another embodiment, the carrier is a biodegradable biocompatiblepolyketal wherein at least a subset of the polyketal repeat structuralunits have the following chemical structure:

-   -   wherein each occurrence of R¹ and R² is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation. In certain embodiments, the carbonyl group        is an aldehyde moiety.

Examples of suitable organic moieties include, but are not limited to,aliphatic groups having a chain of atoms in a range of between about oneand twelve atoms, hydroxyl, hydroxyalkyl, amine, carboxyl, amide,carboxylic ester, thioester, aldehyde, nitryl, isonitryl, nitroso,hydroxylamine, mercaptoalkyl, heterocycle, carbamates, carboxylic acidsand their salts, sulfonic acids and their salts, sulfonic acid esters,phosphoric acids and their salts, phosphate esters, polyglycol ethers,polyamines, polycarboxylates, polyesters, polythioesters,pharmaceutically useful groups, a biologically active substance or adiagnostic label.

Structure, yield and molecular weight of the resultant polyaldehyde(i.e., polyal) depend on the initial polysaccharide. Polysaccharidesthat do not undergo significant depolymerization in the presence ofglycol-specific oxidizing agents, for example, poly(2,1) and (2, 6)fructoses, are preferable. Examples of suitable polysaccharides includealpha and beta 2,1 and 2,6 fructans. Other exemplary polysaccharidesinclude Inulin, Levans from plants, and bacterial fructans. Examples ofsuitable glycol-specific oxidizing agents include sodium periodate, leadtetra-acetate, periodic acid, etc. In certain embodiments, the oxidationsystem consists of a non-specific oxidizing agent in combination withglycol-specific catalyst or and intermediate oxidizer, or anelectrochemical cell. Examples of suitable reducing agents includesodium borohydride, sodium cyanoborohydride, etc. Temperature, pH andreaction duration can affect the reaction rate and polymer hydrolysisrate. The reaction is preferably conducted in the absence of light. Oneskilled in the art can optimize the reaction conditions to obtainpolymers of desired composition. The resultant polymeric aldehydeintermediate may be reacted with suitable H₂N—O-containing modifiermoieties to generate conjugates comprising one or more modifierscovalently attached to the carrier via oxime linkages, as described inmore detail below.

In certain embodiments, under physiological conditions, at least one ofthe aldehyde groups in the aldehyde-substituted polyal can exist in ahydrated (hem-diol) form. As such, the aldehyde group is considered ahydrophilic group. In another embodiment, the precursor carbohydrate hasa chiral atom outside of the cleavage site. Thus the chirality of thatatom is retained, and the polyal is chiral or optically active.

In certain embodiments, the polyals of the present invention can containintermittent irregularities throughout the polyals, such as incompletelyoxidized additional groups or moieties in the main chain or in the sidechains.

Although it is generally understood that each acetal/ketal unit in apolyal of the present invention can have different R¹, R², R³, R⁴, R⁵and R⁶ groups, in certain exemplary embodiments, more than 50% of theacetal/ketal units have the same R¹, R², R³, R⁴, R⁵, and R⁶. Forexample, exemplary polyals for use in this invention include polymers ofthe general formula:

-   -   where n is an integer from 1-5000.

Since it is believed that oxidation does not affect configurations at C¹and C², the polyal retain the configuration of the parentpolysaccharide. Thus, the polyals (and corresponding conjugates) can beformed in stereoregular isotactic forms.

Fully Synthetic Route

In another preferred embodiment, the biodegradable biocompatible polyalsof the present invention can be prepared by reacting a suitableinitiator with a suitable precursor compound, as described, for examplein U.S. Pat. Nos. 5,811,510; 5,863,990 and 5,958,398; U.S. ProvisionalPatent Application 60/348,333; European Patent No.: 0820473; andInternational Patent Application PCT/US03/01017.

For example, fully synthetic polyals may be prepared by condensation ofvinyl ethers with protected substituted diols. Other methods, such ascycle opening polymerization, may be used, in which the method efficacymay depend on the degree of substitution and bulkiness of the protectivegroups.

One of ordinary skill in the art will appreciate that solvent systems,catalysts and other factors may be optimized to obtain high molecularweight products.

Modifiers

As discussed above, modifiers useful in the practice of the inventionmay be adapted for covalent binding with a carbonyl group present on acarrier via an oxime linkage. For example, a modifier may be covalentlyattached to a linker moiety comprising a functional group that can forman oxime linkage with a carbonyl group present on a carrier. Forinstance, the inventive modifiers may have the structure:

-   -   wherein M is a modifier; L^(M1) is a substituted or        unsubstituted, cyclic or acyclic, linear or branched        C₀₋₁₂alkylidene or C₀₋₁₂alkenylidene moiety wherein up to two        non-adjacent methylene units are independently optionally        replaced by CO, CO₂, COCO, CONR^(Z1), OCONR^(Z1),        NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,        NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1),        NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein each occurrence of        R^(Z1) and R^(Z2) is independently hydrogen, alkyl, heteroalkyl,        aryl, heteroaryl or acyl; and R^(N1) and R^(N2) are        independently hydrogen, an aliphatic, alicyclic,        heteroaliphatic, heterocyclic, aryl or heteroaryl moiety, or a        nitrogen protecting group, or R^(N1) and R^(N2), taken together        with the nitrogen atom to which they are attached, form a        substituted or unsubstituted heterocyclic or heteroaryl moiety.

In certain embodiments, the above-described modifiers may be prepared bynucleophilic addition of an aminooxy reagent (i) with a suitablemodifier-containing reagent (ii), as shown below:

-   -   wherein Y is a suitable leaving group.

In certain embodiments, the modifier-containing reagent (ii) may beprepared by nucleophilic addition of a modifier M with a suitable linker(iii), as shown below:

-   -   wherein L¹ is an aliphatic, alicyclic, heteroaliphatic,        heterocyclic, aryl or heteroaryl moiety comprising a functional        group adapted for covalent binding to the modifier.

In certain other embodiments, the above-described modifiers may beprepared by nucleophilic addition of an aminooxy reagent (i) with asuitable linker (iii), followed by reaction of the resulting adduct (iv)with a suitable modifier M, as shown below:

-   -   wherein Y is a suitable leaving group; and L¹ is an aliphatic,        alicyclic, heteroaliphatic, heterocyclic, aryl or heteroaryl        moiety comprising a functional group adapted for covalent        binding to the modifier.

In certain exemplary embodiments, L¹ is a moiety having the structure—(CR^(L1)R^(L2))_(p)-Q-, wherein p is an integer from 0-6, R^(L1) andR^(L2) are independently hydrogen, an aliphatic, alicyclic,heteroaliphatic, heterocyclic, aryl or heteroaryl moiety or WR^(W1)wherein W is O, S, NH, CO, SO₂, COO, CONH, and R^(W1) is hydrogen, analiphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl,alkylaryl or alkylheteroaryl moiety, and Q is a moiety comprising afunctional group adapted for covalent binding to a modifier. In certainother exemplary embodiments, L¹ is —(CH₂)_(p) wherein p is an integerfrom 0-5. In yet other exemplary embodiments, L¹ is—CH₂)_(p1)—CH(OH)CH₂NH— wherein p₁ is an integer from 1-5.

In certain embodiments, Q comprises an active ester moiety useful forconjugation with amino groups. In certain exemplary embodiments, Qcomprises N-hydroxy succinimide ester, tetrafluorophenyl ester ornitrophenyl ester. In certain other exemplary embodiments, Q comprises asuccinimidyl ester moiety having the structure:

In certain embodiments, Q comprises a maleimido moiety useful forconjugation with thio groups. In certain other exemplary embodiments, Qcomprises a succinimidyl ester moiety having the structure:

In certain other exemplary embodiments, L¹ is —(CH₂)_(p) wherein p is aninteger from 0-5; and Q a maleimidyl moiety having the structure:

-   -   wherein p₂ is an integer from 1-5.

In yet other exemplary embodiments, L¹ is —(CH₂)_(p1)—CH(OH)CH₂NH—wherein p₁ is an integer from 1-5, and Q is a maleimidyl moiety havingthe structure:

-   -   wherein p₂ is an integer from 1-5.

In certain embodiments, one of R^(N1) and R^(N2) is a nitrogenprotecting group. Nitrogen protecting groups, as well as protection anddeprotection methods are known in the art. Guidance may be found in“Protective Groups in Organic Synthesis” Third Ed. Greene, T. W. andWuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entirecontents of which are hereby incorporated by reference.

In certain exemplary embodiments, R^(N1)R^(N2)N— is a moiety having thestructure:

In yet other exemplary embodiments, L¹ is —CH₂)_(p1)—CH(OH)CH₂NH-Qwherein p₁ is an integer from 1-5; R^(N1)R^(N2)N— is a moiety having thestructure:

-   -   wherein Q is a moiety comprising a functional group adapted for        covalent binding to a modifier, and the invention provides        compounds having the structure:

In certain exemplary embodiments, the compounds described directly abovemay be prepared by reaction of oxime (v) with epichlorohydrine (vi) toform the corresponding epoxide adduct (vii), followed by reacting (vii)with a suitable nucleophile R—ZH to give adduct (viii), wherein Z is anucleophilic atom (e.g., O, S, NH) and R is hydrogen, an organic moietycomprising Q, or an organic moiety that can be modified to incorporateQ.

Oxime (viii) can deprotected in mild acidic conditions to givebifunctional compound (ix):

In certain embodiments, R comprises an active ester moiety useful forconjugation with amino groups. In certain exemplary embodiments, Rcomprises N-hydroxy succinimide ester, tetrafluorophenyl ester ornitrophenyl ester. In certain other exemplary embodiments, R comprises asuccinimidyl ester moiety having the structure:

In certain embodiments, R comprises a maleimido moiety useful forconjugation with thio groups. In certain other exemplary embodiments, Rcomprises a succinimidyl ester moiety having the structure:

In certain embodiments, R is a carboxyl. In another embodiment, R is asecond aminooxy group.

Conjugates

In another aspect, the invention provides a method for preparing aconjugate comprising a carrier substituted with one or more occurrencesof a moiety having the structure:

-   -   wherein each occurrence of M is independently a modifier; and    -   each occurrence of L^(M) is independently an oxime-containing        linker;    -   said method comprising steps of:    -   providing a carrier;    -   providing one or more modifiers;    -   providing one or more compounds having the structure:        R^(N1)R^(N2)N—O-L¹; wherein R^(N1) and R^(N2) are independently        hydrogen, an aliphatic, alicyclic, heteroaliphatic,        heterocyclic, aryl or heteroaryl moiety, or a nitrogen        protecting group, or R^(N1) and R^(N2), taken together, form a        substituted or unsubstituted alicyclic, aryl or heteroaryl        moiety; and each occurrence of L¹ is independently an aliphatic,        alicyclic, heteroaliphatic, heterocyclic, aryl or heteroaryl        moiety comprising a functional group adapted for covalent        binding to the modifier; and    -   reacting the one or more compounds of structure        R^(N1)R^(N2)N—O-L¹ with the carrier and the one or more        modifiers under suitable conditions so that at least one        —O—NR^(N1)R^(N2) moiety is covalently attached to the carrier        via an oxime linkage, thereby generating the conjugate.

In another aspect, the invention provides a method for preparing aconjugate comprising a carrier substituted with one or more occurrencesof a moiety having the structure:

-   -   wherein each occurrence of M is independently a modifier; and    -   each occurrence of L^(M) is independently an oxime-containing        linker;    -   said method comprising steps of:    -   providing a carrier;    -   providing one or more compounds having the structure:

-   -   wherein L^(M1) is a substituted or unsubstituted, cyclic or        acyclic, linear or branched C₀₋₁₂alkylidene or C₀₋₁₂alkenylidene        moiety wherein up to two non-adjacent methylene units are        independently optionally replaced by CO, CO₂, COCO, CONR^(Z1),        OCONR^(Z1), NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO,        NR^(Z1)CO₂, NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1),        NR^(Z1)SO₂NR^(Z2), O, S, or NR^(Z1); wherein each occurrence of        R^(Z1) and R^(Z2) is independently hydrogen, alkyl, heteroalkyl,        aryl, heteroaryl or acyl; and R^(N1) and R^(N2) are        independently hydrogen, an aliphatic, alicyclic,        heteroaliphatic, heterocyclic, aryl or heteroaryl moiety, or a        nitrogen protecting group, or R^(N1) and R^(N2), taken together        with the nitrogen atom to which they are attached, form a        substituted or unsubstituted heterocyclic or heteroaryl moiety;        and    -   reacting the carrier with the one or more compounds of        structure:

-   -   under suitable conditions so that at least one —O—NR^(N1)R^(N2)        moiety is covalently attached to the carrier via an oxime        linkage, thereby generating the conjugate.

In certain exemplary embodiments, R^(N1) and R^(N2) are each hydrogen.In certain exemplary embodiments, in the one or more compounds ofstructure R^(N1)R^(N2)N—O-L¹; at least one of R^(N1) and R^(N2) is anitrogen protecting group; and the method further comprises the step ofhydrolyzing the one or more compounds having the structureR^(N1)R^(N2)N—O-L¹ to form one or more compounds having the structureH₂N—O-L¹ prior to reacting with the carrier. In certain exemplaryembodiments, in the one or more compounds of structureR^(N1)R^(N2)N—O-L¹, R^(N1)R^(N2)N— has the structure CH₃CH₂OC(CH₃)═N—;and the one or more compounds have the following structure:

In certain exemplary embodiments, in the one or more compounds ofstructure

at least one of R^(N1) and R^(N2) is a nitrogen protecting group; andthe method further comprises the step of hydrolyzing the one or morecompounds having the structure:

-   -   to form one or more compounds having the structure:

-   -   prior to reacting with the carrier.

In certain exemplary embodiments, in the one or more compounds ofstructure:

R^(N1)R^(N2)N— has the structure CH₃CH₂OC(CH₃)═N—; and the one or morecompounds have the following structure:

In certain exemplary embodiments, in practicing the method of theinvention, the carrier is a biodegradable biocompatible polyacetalwherein at least a subset of the polyacetal repeat structural units havethe following chemical structure:

-   -   wherein for each occurrence of the n bracketed structure, one of        R¹ and R² is hydrogen, and the other is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation. In certain embodiments, the carbonyl group        is an aldehyde moiety.

In certain exemplary embodiments, in practicing the method of theinvention, the carrier is a biodegradable biocompatible polyketalwherein at least a subset of the polyketal repeat structural units havethe following chemical structure:

-   -   wherein each occurrence of R¹ and R² is a biocompatible group        and includes a carbon atom covalently attached to C¹; R^(x)        includes a carbon atom covalently attached to C²; n is an        integer; each occurrence of R³, R⁴, R⁵ and R⁶ is a biocompatible        group and is independently hydrogen or an organic moiety; and        for each occurrence of the bracketed structure n, at least one        of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitable        for oxime formation. In certain embodiments, the carbonyl group        is an aldehyde moiety.

Bifunctional compounds R^(N1)R^(N2)N—O-L¹ and modifiers of thestructure:

-   -   of both protected (i.e., R^(N1)R^(N2)N— is a protected nitrogen        moiety) and deprotected (i.e., R^(N1) and R^(N2) are each        hydrogen) types can be used in conjugation. The latter may be        performed in a variety of ways. For example, a protected or        deprotected maleimido-aminooxy reagent can be first conjugated        with thiol groups present in a modifier (e.g., in a protein or        peptide). If a protected form of reagent was used, the        protection can be removed under conditions suitable to remove        the selected nitrogen protecting group. For example,        R^(N1)R^(N2)N— is a moiety having the structure:

-   -   nitrogen deprotection can be effected, in aqueous media at pH<7,        preferably from 2 to 4. Then, the resultant aminooxyderivative        can be conjugated with a carbonyl-comprising carrier in aqueous        media at pH 3 to 6, preferably 4 to 5. Conjugation can be        performed using a mixture of bifunctional compounds        R^(N1)R^(N2)N—O-L¹ and/or modifiers of the structure:

Conversion (conjugation) degree can be monitored by any suitable method,for example HPLC. When the desirable conversion degree has beenachieved, the product can be purified (e.g., by gel chromatography) andisolated (e.g., via lyophilization). Conversely, the bifunctionalreagent can be first reacted with a carbonyl-comprising carrier, andthen the “activated” product can be reacted with the respectivemodifier.

To obtain a conjugate containing a set of different modifiers,conjugation can be performed in one step in a mixture ofaminooxy-containing modifiers having the structure:

The latter can be synthesized, for example, as described above. Then,all modifiers can be mixed with the carbonyl-containing carrier and thereaction mixture incubated in the described conditions until thedesirable conversion degree is achieved. This method can be used, viamixing the modifiers and the carrier at different ratios, to produce, inone step, libraries of conjugates with varying modifier composition andcontent.

In either one of the above methods, the residual unreacted aminooxybifunctional reagent and/or modifier, as well as unreacted carbonylgroups on the carrier can be “quenched”, if desired, with a suitablereagent. For example, carbonyl groups can be either oxidized intocarboxyls (e.g., with iodine), or reacted with a suitableaminooxy-substituted compound, e.g., 1-aminooxy-propanediol-2,3.Unreacted aminooxygroups can be reacted with a suitable aldehyde orketone (e.g., acetaldehyde).

Compositions

In certain embodiments, there is provided a composition comprising anyone or more of the conjugates disclosed herein and a pharmaceuticallysuitable carrier or diluent.

In certain embodiments, the invention provides a composition in the formof a gel of the inventive biodegradable biocompatible polyal conjugateand a biologically active compound disposed within the gel.Alternatively or additionally, a diagnostic label can be disposed withinthe gel or bound to the gel matrix.

In another embodiment, the invention provides a composition in the formof a solution of the biodegradable biocompatible polyal conjugate and apharmaceutically useful entity, a drug or a macromolecule dissolvedwithin the solution. Alternatively or additionally, a diagnostic labelcan be dissolved within the solution.

In certain embodiments, there is provided a composition comprising abiodegradable biocompatible polyal conjugate of the invention associatedwith an effective amount of a therapeutic agent; wherein the therapeuticagent is incorporated into an released from said biodegradablebiocompatible polyal conjugate matrix by degradation of the polymermatrix or diffusion of the agent out of the matrix over a period oftime. In certain embodiments, polyal conjugate is non-covalentlyassociated with an effective amount of a therapeutic agent. In certainembodiments, the therapeutic agent is selected from the group consistingof vitamins, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents, and combination thereof.

In variations of these embodiments, it may be desirable to include otherpharmaceutically active compounds, such as antiinflammatories orsteroids which are used to reduce swelling, antibiotics, antivirals, orantibodies. Other compounds which can be included are preservatives,antioxidants, and fillers, coatings or bulking agents which may also beutilized to alter polymer matrix stability and/or drug release rates.

Additives Used to Alter Properties of Conjugate Compositions:

In a preferred embodiment, only polyal conjugate and drugs to bereleased are incorporated into the delivery device or construct,although other biocompatible, preferably biodegradable or metabolizable,materials can be included for processing, preservation and otherpurposes.

Buffers, acids and bases are used to adjust the pH of the composition.Agents to increase the diffusion distance of agents released from theimplanted polymer can also be included.

Fillers are water soluble or insoluble materials incorporated into theformulation to add bulk. Types of fillers include sugars, starches andcelluloses. The amount of filler in the formulation will typically be inthe range of between about 1 and about 90% by weight.

Methods of Use

The present invention encompasses polymer conjugates for use inbiomedical applications, primarily (but not exclusively) in the fieldsof pharmacology, bioengineering, wound healing, anddermatology/cosmetics. In certain embodiments, the polymer conjugatesare biodegradable polyal conjugates. In particular, medical applicationsfor the conjugates of the invention include tablet coatings, plasmasubstitutes, gels, contact lenses, surgical implants, systems forcontrolled drug release, as ingredients of eyedrops, wound closureapplications (sutures, staples), orthopedic fixation devices (pins,rods, screws, tacks, ligaments), dental applications (guided tissueregeneration), cardiovascular applications (stents, grafts), intestinalapplications (anastomosis rings), and as long circulating and targeteddrugs. Conjugates of the present invention can be employed as componentsof biomaterials, drugs, drug carriers, pharmaceutical formulations,medical devices, implants, and can be associated with small molecules,pharmaceutically useful entities, drugs, macromolecules and diagnosticlabels.

Methods of Treating

In certain preferred embodiments of the invention, the conjugates of theinvention are used in methods of treating animals (preferably mammals,most preferably humans). In one embodiment, the conjugates of thepresent invention may be used in a method of treating animals whichcomprises administering to the animal a biodegradable biocompatibleconjugates of the invention. For example, conjugates in accordance withthe invention can be administered in the form of soluble linearpolymers, copolymers, conjugates, colloids, particles, gels, soliditems, fibers, films, etc. Biodegradable biocompatible conjugates ofthis invention can be used as drug carriers and drug carrier components,in systems of controlled drug release, preparations for low-invasivesurgical procedures, etc. Pharmaceutical formulations can be injectable,implantable, etc.

In yet another aspect, the invention provides a method of administeringto a patient in need of treatment, comprising administering to thesubject an effective amount of a suitable therapeutic agent; whereinsaid therapeutic agent is associated with and released from a conjugateof the invention by degradation of the conjugate matrix or diffusion ofthe agent out of the matrix over a period of time.

In certain embodiments, the therapeutic agent is locally delivered byimplantation of said conjugate matrix incorporating the therapeuticagent.

In certain embodiments, the therapeutic agent is selected from the groupconsisting of: vitamins, anti-AIDS substances, anti-cancer substances,antibiotics, immunosuppressants, anti-viral substances, enzymeinhibitors, neurotoxins, opioids, hypnotics, anti-histamines,lubricants, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmodics and muscle contractantsincluding channel blockers, miotics and anti-cholinergics, anti-glaucomacompounds, anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents, and combinations thereof.

In certain other exemplary embodiments, the method further comprisesadministering with the therapeutic agent additional biologically activecompounds selected from the group consisting of vitamins, anti-AIDSsubstances, anti-cancer substances, antibiotics, immunosuppressants,anti-viral substances, enzyme inhibitors, neurotoxins, opioids,hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants,muscle relaxants and anti-Parkinson substances, anti-spasmodics andmuscle contractants including channel blockers, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, vasodilating agents, inhibitors of DNA, RNA or proteinsynthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal andnon-steroidal anti-inflammatory agents, anti-angiogenic factors,anti-secretory factors, anticoagulants and/or antithrombotic agents,local anesthetics, ophthalmics, prostaglandins, anti-depressants,anti-psychotic substances, anti-emetics, imaging agents, and combinationthereof.

In certain embodiments, in practicing the method of the invention, theconjugate further comprises or is associated with a diagnostic label. Incertain exemplary embodiments, the diagnostic label is selected from thegroup consisting of: radiopharmaceutical or radioactive isotopes forgamma scintigraphy and PET, contrast agent for Magnetic ResonanceImaging (MRI), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves and fluorophores. Incertain exemplary embodiments, the conjugate is further monitored invivo.

In another aspect, the invention provides a method of administering aconjugate of the invention to an animal, comprising preparing an aqueousformulation of said conjugate and parenterally injecting saidformulation in the animal. In certain exemplary embodiments, theconjugate comprises a biologically active modifier. In certain exemplaryembodiments, the conjugate comprises a detectable modifier.

In another aspect, the invention provides a method of administering aconjugate of the invention to an animal, comprising preparating animplant comprising said conjugate, and implanting said implant into theanimal. In certain exemplary embodiments, the implant is a biodegradablegel matrix.

In another aspect, the invention provides a method for treating of ananimal in need thereof, comprising administering a conjugate accordingto the methods described above, wherein said conjugate is associatedwith a biologically active component.

In another aspect, the invention provides a method for treating of ananimal in need thereof, comprising administering a conjugate accordingto the methods described above, wherein said conjugate comprises abiologically active modifier. In certain exemplary embodiments, thebiologically active component is a gene vector.

In another aspect, the invention provides a method for eliciting animmune response in an animal, comprising administering a conjugate as inthe methods described above, wherein said conjugate comprises an antigenmodifier.

In another aspect, the invention provides a method of diagnosing adisease in an animal, comprising steps of:

-   -   administering a conjugate as in the methods described above,        wherein said conjugate comprises a detectable modifier; and    -   detecting the detectable modifier.

In certain exemplary embodiments, the step of detecting the detectablemodifier is performed non-invasively. In certain exemplary embodiments,the step of detecting the detectable modifier is performed usingsuitable imaging equipment.

In one embodiment, a method for treating an animal comprisesadministering to the animal the biodegradable biocompatible conjugatesof the invention as a packing for a surgical wound from which a tumor orgrowth has been removed. The biodegradable biocompatible conjugatepacking will replace the tumor site during recovery and degrade anddissipate as the wound heals.

In certain embodiments, the conjugate is associated with a diagnosticlabel for in vivo monitoring.

The conjugates described above can be used for therapeutic,preventative, and analytical (diagnostic) treatment of animals. Theconjugates are intended, generally, for parenteral administration, butin some cases may be administered by other routes.

In one embodiment, soluble or colloidal conjugates are administeredintravenously. In another embodiment, soluble or colloidal conjugatesare administered via local (e.g., subcutaneous, intramuscular)injection. In another embodiment, solid conjugates (e.g., particles,implants, drug delivery systems) are administered via implantation orinjection.

In one embodiment, conjugates comprising a biologically active substance(e.g., a drug or a gene vector) are administered to treat diseaseresponsive to said substance.

In another embodiment, conjugates comprising a detectable label areadministered to study the patterns and dynamics of label distribution inanimal body.

In another embodiment, conjugates comprising an antigen or anantigen-generating component (e.g., a plasmid) are administered todevelop immunity to said antigen.

Applications to Drug Delivery Methods

Polyal-small-molecule-drug conjugates: In one embodiment, pharmaceuticalagents are associated with the biodegradable biocompatible conjugate ofthe invention to form a biodegradable biocompatible gel or mass ofconjugate in which the drug is entrapped or bound to gel matrix, or asoluble conjugate of a drug and a polyal conjugate. This can beachieved, for example, by coupling the conjugate of the invention with adrug modifier via oxime-containing linkages (for example, taxol orcamptothecin (CPT)). Alternatively, the drug can be entrapped bydissolution of the drug in the presence of the biodegradablebiocompatible conjugate during removal of a solvent, or duringcrosslinking. When soluble polyal-drug conjugates are administered(e.g., injected) into an animal, they can circulate and accumulate at adesirable site, and slowly release the drug either in circulation, or atthe accumulation site, either intracellularly or extracellularly. Whengels or masses are implanted into an animal, slow hydrolysis of thebiodegradable biocompatible conjugate mass or gel occurs with continuousslow release of the agent in the animal at the location where itsfunction is required. Such polymer-drug pharmaceutical compositions mayafford release of the physiologically active substance intophysiological fluids in vivo over a sustained period (for an example ofpolymer-drug conjugate, see Li, et al. “Water soluble paclitaxelprodrugs” U.S. Pat. No. 6,262,107, 2001, the entire contents of whichare incorporated herein by reference). In addition, the hydrophilicconjugates of the invention may be used to stabilize drugs, as well asto solubilize otherwise insoluble compounds. For example, Paclitaxel, ananti-microtubule agent that has shown a remarkable anti-neoplasticeffect in human cancer in Phase I studies and early Phase II and IIItrials (Horwitz et al., “Taxol, mechanisms of action and resistance,” J.Natl. Cancer Inst. Monographs No. 15, pp. 55-61, 1993), has limitedsolubility in water, which has hampered its development for clinicaltrial use. The polyal-drug conjugate pharmaceutical compositions of theinvention could provide water soluble taxoids to overcome the drawbacksassociated with the insolubility of the drugs themselves, and alsoprovide the advantages of accumulation in tumors, targeting to cancercells and controlled release so that tumors may be eradicated moreefficiently. Association of chemotherapeutic drugs to the conjugates ofthe invention may also be an attractive approach to reduce systemictoxicity and improve the therapeutic index. In particular, it is knownin the art that polymers with molecular mass larger than 30 kDa do notreadily diffuse through normal capillaries and glomerular endothelium,thus sparing normal tissue from irrelevant drug-mediated toxicity (Maedaand Matsumura, “Tumoritropic and lymphotropic principles ofmacromolecular drugs”, Critical Review in Therapeutic Drug CarrierSystems, 6:193-210, 1989; Reynolds, T., “Polymers help guide cancerdrugs to tumor targets—and keep them there,” J. Natl. Cancer Institute,87:1582-1584, 1995). On the other hand, it is well established thatmalignant tumors often have altered capillary endothelium and greaterpermeability than normal tissue vasculature (Maeda and Matsumura, 1989;Fidler, et al., “The biology of cancer invasion and metastasis,” Adv.Cancer Res., 28:149-250, 1987). Thus, a polymer-drug conjugate, such asthose described in the present invention, that would normally remain inthe vasculature, may selectively leak from blood vessels into tumors,resulting in tumor accumulation of active therapeutic drug. The methodsdescribed herein could also be used to make water soluble conjugatecomplexes of other therapeutic agents, contrast agents and drugs.

protein-modified carriers: In certain embodiments, carriers may beassociated to a protein or peptide (for example enzymes or growthfactors) to form a protein/peptide-modified conjugate. Improved chemicaland genetic methods have made many enzymes, proteins, and other peptidesand polypeptides available for use as drugs or biocatalysts havingspecific catalytic activity. However, limitations exist to the use ofthese compounds. For example, enzymes that exhibit specific biocatalyticactivity sometimes are less useful than they otherwise might be becauseof problems of low stability and solubility. During in vivo use, manyproteins are cleared from circulation too rapidly. Some proteins haveless water solubility than is optimal for a therapeutic agent thatcirculates through the bloodstream. Some proteins give rise toimmunological problems when used as therapeutic agents. Immunologicalproblems have been reported from manufactured proteins even where thecompound apparently has the same basic structure as the homologousnatural product. The use of protein/peptide-modified conjugates may helpprotect the protein/peptide from chemical attack, limit its adverse sideeffects when injected into the body, increase its size, and may thuspotentially improve its therapeutic profile in vivo (e.g., safety,efficacy and stability in biological media). See for example Harris etal. “Multiarmed, monofunctional, polymer for coupling to molecules andsurfaces” U.S. Pat. No. 5,932,462, 1999. Examples of proteins that maybe used in this context are enzymes, recognition proteins, carrierproteins, and signaling proteins and polypeptides, such as, urokinase,catalase, hemoglobin, granulocyte colony stimulating factor (G-CSF),interferons, cytokines, leptins, insulin, etc.

Although there is no theory that predicts the optimal composition, sizeand shape of a macromolecule conjugate, it can be expected that, forsome applications, conjugates consisting of one protein molecule and onepolyal molecule will be desirable, whereas in other applicationsconjugates comprising several identical or different protein or peptidemolecules per polyal molecule can be preferable. In one preferredembodiment, a protein is conjugated with a polyal of the invention via aterminal group of the latter. In another embodiment, one or more proteinor peptide molecules are conjugated to the polyal molecule of theinvention at random points.

Cationized polyal: In another embodiment, the polyals of the presentinvention may find use as a nucleic acid carrier vehicle for delivery ofnucleic acid material to target cells in biological systems (for examplein applications using adducts with DNA or Polyal-modified virus). Suchmaterial may find applications for in vivo delivery of genes ortherapeutic DNA to a patient in carrying out gene therapy or DNAvaccination treatment (See for example Schacht et al. “Delivery ofnucleic acid material” U.S. Pat. No. 6,312,727, 2001; German et al.“Enhanced adenovirus-assisted transfection composition and method” U.S.Pat. No. 5,830,730, 1998). For example, the polyal may be synthesized ormodified so as to form a “cationized” material whereby one or morecationic sites are included or incorporated in the polyal molecule.Association or binding of this cationized hydrophilic polymer with apolyanionic nucleic acid component results in a material that mayfunction as a DNA or nucleic acid delivery device. The nucleic acidcomponent may comprise a polynucleotide, plasmid DNA, lineardouble-helical DNA, RNA or a virus. In another embodiment, the cationicpolyal core may be associated, directly or indirectly, to othermolecular entities or moieties, especially bioactive molecules, thatmodify the biological and/or physico-chemical characteristics of thecomplex to improve suitability or specificity for use in delivering thenucleic acid material to target cells. These other molecular entities ormoieties may comprise cell-receptor targeting moieties and/or otherspecific bioactive agents providing, for example, membrane disruptingagents, agents capable of promoting endocytic internalization followingbinding to cell surface molecules, and nuclear-homing agents, useful forfacilitating entry and delivery of the nucleic acid material, e.g. DNA,into cells.

Polyal-modified liposomes: In yet another embodiment, the polyals of thepresent invention may be associated with a liposome (see for exampleDadey “Polymer-associated liposomes for drug delivery and method ofmanufacturing the same” U.S. Pat. No. 5,935,599, 1999). In certainembodiments, the polyal-associated liposome is formulated with a drug ora therapeutic agent to provide a drug composition that treats anunderlying disease or complications associated with the disease. Thepolyal-associated liposome may be formulated with either water-solubleor water-insoluble drugs, or both. Therefore, a drug compositioncontaining a polyal-associated liposome and a drug can be administeredin a variety of dosage forms. A liposome is a mono- or multilamellarvesicle prepared from a phospholipid or other suitable lipids ormixtures thereof. Structurally, lamellae are bilayer membranes havingpolar ends of lipids in one layer forming the external surface of thespherical membrane and the polar ends of lipids in a second layerforming the internal surface of the spherical membrane. Membranes caninclude hydrophobic additives, such as cholesterol. The nonpolar,hydrophobic tails of the lipids in the two layers self-assemble to formthe interior of the bilayer membrane. Liposomes can microencapsulatecompounds, and transport the compounds through environments wherein theyare normally degraded. The liposome can be prepared by conventionaltechniques from phosphatidylethanolamine, phosphatidylcholine,phosphatidylserine, phosphatidylinositol, phostidylglycerol,sphingomyelin, and mixtures thereof. The outer layer of a liposome canbe modified with a polyal to either prevent liposome aggregation, or toprolong liposome circulation in blood, or for other purposes.Preferably, polyal molecules are chemically linked to lipid moleculesconstituting the outer membrane. In certain embodiments, polyalmolecules are chemically linked to lipid molecules constituting theouter membrane via oxime linkages. Some or all polyal molecules can befurther modified with targeting moieties that assist liposome binding totarget cells or tissues. In a preferred embodiment, polyal molecules arelinked to lipid molecules through terminal groups, forming lipid-polyalconjugates. The latter can be incorporated into liposomes during theprocess of liposome formation, e.g. by extrusion. Alternatively, polyalscan be chemically bound to pre-formed liposomes comprising suitablefunctional groups on the outer surface (e.g., amino, mercapto, orcarboxygroups).

Polyal-modified nano- and microparticles: In a further embodiment of thepresent invention, the polyal conjugates of the invention may bedesigned so as to have properties suitable for manufacturing by variousprocesses into nanoparticles, microparticles and microspheres forapplications in drug delivery systems. Polyal conjugates can be utilizedin such applications as interface components, particle matrixcomponents, or both. Where polyal conjugates are used as interfacecomponents, the (inner) particle can be a nanoparticle (e.g., iron oxidenanocrystal or combination thereof), a latex particle (e.g., polystyrenenanosphere or microsphere), a gel particle (e.g., crosslinked polyketal,polyacetal or polysaccharide gel sphere), etc. Where the polyalconjugate is used as a matrix component, alone or along with othermacromolecular components or particulates, the polytal molecules can bechemically crosslinked or non-chemically associated to form a gel or asolid, and can be chemically or physically associated with a drug. Thelatter becomes, therefore, incorporated or entrapped in the particle,and can subsequently be released via diffusion or degradationmechanisms.

The slow-release characteristic of the polymer microparticles may alsohave use in the field of pharmacology where the microparticles can beused, for example, to deliver pharmacological agents in a slow andcontinual manner (see for example Sokoll et al. “Biodegradabletargetable microparticle delivery system” U.S. Pat. No. 6,312,732,2001). A wide range of drugs such as anti-hypertensives, analgesics,steroids and antibiotics can be used in accordance with the presentinvention to provide a slow release drug delivery system. Largemolecules, such as proteins, can also be entrapped in micro- andnanoparticles, using methods of particle formation that do notinactivate the large molecule. Microspheres may be prepared by knownmethods in the art, for example, using a single emulsification process(U.S. Pat. No. 4,389,330 to Tice et al.; U.S. Pat. No. 3,691,090 toKitajima et al.), a double emulsification process (Edwards et al.,Science 276: 1868-1871, 1997), a phase inversion microencapsulationprocess (Mathiowitz et al., Nature 386: 410-413, 1997), or anatomization-freeze process (Putney and Burke, Nature Biotechnology 16:153-157, 1998). In the single emulsification process, a volatile organicsolvent phase containing a biodegradable polymer, an aqueous solutioncontaining an emulsifier such as polyvinyl alcohol, and aphysiologically active substance are homogenized to produce an emulsion.The solvent is evaporated and the resulting hardened microspheres arefreeze-dried. In the double emulsification process, an aqueous solutionwhich may contain a physiologically active substance and a volatileorganic solvent phase containing a biodegradable polymer are homogenizedto form an emulsion. The emulsion is mixed with another aqueoussolution, which contains an emulsifier such as polyvinyl alcohol.Evaporation of the solvent and freeze-drying produces microspheres. Inthe phase inversion microencapsulation process, the drug is added to adilute polymer solution in a solvent (e.g. dichloromethane) which isthen poured rapidly into an unstirred bath of another liquid (e.g.petroleum ether) causing nano- and microspheres to form spontaneously.In the atomization-freeze process, the micronized solid physiologicallyactive substance is suspended in a solvent phase containing abiodegradable polymer that is then atomized using sonication orair-atomization. This produces droplets that are then frozen in liquidnitrogen. Addition of another solvent in which both the polymer and thedrug are insoluble extracts the solvent from the microspheres. In suchprocesses, polyal conjugates can be used as interface components formedduring or after particle formation. Preferably, the process isengineered such that polyal conjugates form a monolayer on the particlesurface, which is dense enough to modify the particle surfacehydrophilicity, and/or to prevent direct contact of cells and/orrecognition proteins with the particle surface. This can be achieved,for example, by chemical coupling of the polyal to the surface of thepore-formed particles, or through addition of polyal-matrix polymerconjugates into technological solutions. Such conjugates (e.g., blockcopolymers) will, in appropriately optimized conditions, incorporateinto particles such that the matrix polymer block will incorporate intothe particle body, while the polyal conjugate block will be exposed onthe particle surface. Similar approaches can be used for themodification of inorganic particles (such as colloids and nanocrystals)with ketals during or after their formation. Polyal conjugates can beattached to the surfaces of such particles either chemically(conjugation or grafting) or physically (adsorption). A furtherdescription of polyal conjugate use as an interface component is givenin one of the following sections.

In another embodiment, the biodegradable biocompatible polyal conjugatesof the present invention can be monitored in vivo by suitable diagnosticprocedures. Such diagnostic procedures include nuclear magneticresonance imaging (NMR), magnetic resonance imaging (MRI), ultrasound,X-ray, scintigraphy, positron emission tomography (PET), etc. Thediagnostic procedure can detect, for example, polyal conjugatedisposition (e.g., distribution, localization, density, etc.) or therelease of drugs, prodrugs, biologically active compounds or diagnosticlabels from the biodegradable biocompatible polyal conjugate over aperiod of time. Suitability of the method largely depends on the form ofthe administered polyal conjugate and the presence of detectable labels.For example, the size and shape of polyal conjugate implants can bedetermined non-invasively by NMR imaging, ultrasound tomography, orX-ray (“computed”) tomography. Distribution of soluble polyal conjugatepreparation comprising a gamma emitting or positron emitting radiotracercan be performed using gamma scintigraphy or PET, respectively.Microdistribution of polyal conjugate preparation comprising afluorescent label can be investigated using photoimaging.

It is understood, for the purpose of this invention, that transfer anddisposition of polyal conjugates in vivo can be regulated by modifyinggroups incorporated into the polyal conjugate structure, such ashydrophobic and hydrophilic modifiers, charge modifiers, receptorligands, antibodies, etc. Such modification, in combination withincorporation of diagnostic labels, can be used for development of newuseful diagnostic agents. The latter can be designed on a rational basis(e.g., conjugates of large or small molecules binding known tissuecomponents, such as cell receptors, surface antigens, etc.), as well asthrough screening of libraries of polyal conjugate molecules modifiedwith a variety of moieties with unknown or poorly known bindingactivities, such as synthetic peptides and oligonucleotides, smallorganic and metalloorganic molecules, etc.

Interface Component

In one embodiment of the present invention, the biodegradablebiocompatible polyal conjugate can be used as an interface component.The term “interface component” as used herein, means a component, suchas a coating or a layer on an object, to alter the character of objectinteraction with biological interaction with biological milieu, forexample, to suppress foreign body reactions, decrease inflammatoryresponse, suppress clot formation, etc. It should be understood that theobject can be microscopic or macroscopic. Examples of microscopicobjects include macromolecules, colloids, vesicles, liposomes,emulsions, gas bubbles, nanocrystals, etc. Examples of macroscopicobjects include surfaces, such as surfaces of surgical equipment, testtubes, perfusion tubes, items contacting biological tissues, etc. It isbelieved that interface components can, for example, provide the objectprotection from direct interactions with cells and opsonins and, thus,to decrease the interactions of the object with the biological system.

Surfaces can be modified by the biodegradable biocompatible polyalconjugate of the present invention by, for example, conjugatingfunctional groups of the biodegradable biocompatible polyal conjugatewith functional groups present on the surface to be modified. Forexample, aldehyde groups of the biodegradable biocompatible polyalprecursors can be linked with aminooxy groups to form oxime linkages.Alternatively, carboxyl groups of the biodegradable biocompatiblepolyals can be conjugated with amino, hydroxy, sulphur-containinggroups, etc. In another embodiment, a biodegradable biocompatible polyalconjugate of the invention which includes a suitable terminal group canbe synthesized, such as a polyalcohol having a terminal carboxylicgroup. A polymer can be connected to a surface by reaction of theterminal group. Examples of suitable polymers include those formed, forexample, by oxidation of a reducing-end acetal group into a carboxylgroup, such as by using iodine or bromine. The remainder of thepolysaccharide is then oxidized by employing an effective amount of aglycol-specific oxidizing agent to form an aldehyde. The aldehydes canbe selectively modified by, for example, reduction into hydroxyl groups.The resulting polymer will generally have one terminal carboxyl groupthat can be used for one-point modification, such as by employing acarbodiimide.

In still another embodiment, a suitable polysaccharide can be linkedwith a surface by reaction of a reducing or non-reducing end of thepolysaccharide or otherwise, by subsequent oxidation and furtherconversion of the remainder of the polysaccharide to produce a polyalconjugate.

It is to be understood that the biodegradable biocompatible polyalconjugates of this invention can be conjugated with macromolecules, suchas enzymes, polypeptides, proteins, etc., by the methods described abovefor conjugating the biodegradable biocompatible polyal conjugates withfunctional groups present on a surface.

The biodegradable biocompatible polyal conjugates of the invention canalso be conjugated with a compound that can physically attach to asurface via, for example, hydrophobic, van der Waals, and electrostaticinteractions. For example, the biodegradable biocompatible polyalprecursors can be conjugated with lipids, polyelectrolytes, proteins,antibodies, lectins, etc.

It is believed that interface components can prolong circulation ofmacromolecular and colloidal drug carriers. Therefore, small molecules,biologically active compounds, diagnostic labels, etc., beingincorporated in such carriers, can circulate throughout the body withoutstimulating an immunogenic response and without significant interactionswith cell receptors and recognition proteins (opsonins). Further,interface components can be used to modify surfaces of implants,catheters, etc. In other embodiments of the present invention,biomedical preparations of the biodegradable biocompatible polyalconjugates of the invention can be made in various forms. Examplesinclude implants, fibers, films, etc.

Throughout this document, various publications are referred to, each ofwhich is hereby incorporated by reference in its entirety in an effortto more fully describe the state of the art to which the inventionpertains.

The invention will now be further and specifically described by thefollowing examples. All parts and percentages are by weight unlessotherwise stated.

EQUIVALENTS

The representative examples that follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. It should further be appreciated that the contents ofthose cited references are incorporated herein by reference to helpillustrate the state of the art.

The following examples contain important additional information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and the equivalents thereof.

EXEMPLIFICATION

The practitioner has a well-established literature of polymer chemistryto draw upon, in combination with the information contained herein, forguidance on synthetic strategies, protecting groups, and other materialsand methods useful for the synthesis of the conjugates of thisinvention.

The various references cited herein provide helpful backgroundinformation on preparing polymers similar to the inventive compoundsdescribed herein or relevant intermediates, as well as information onformulation, uses, and administration of the conjugates of theinvention, which may be of interest.

Moreover, the practitioner is directed to the specific guidance andexamples provided in this document relating to various exemplaryconjugates and intermediates thereof.

The conjugates of this invention and their preparation can be understoodfurther by the examples that illustrate some of the processes by whichthese compounds are prepared or used. It will be appreciated, however,that these examples do not limit the invention. Variations of theinvention, now known or further developed, are considered to fall withinthe scope of the present invention as described herein and ashereinafter claimed.

According to the present invention, any available techniques can be usedto make or prepare the inventive polyal conjugates or compositionsincluding them. For example, a variety of solution phase syntheticmethods such as those discussed in detail below may be used.Alternatively or additionally, the inventive conjugates may be preparedusing any of a variety combinatorial techniques, parallel synthesisand/or solid phase synthetic methods known in the art.

It will be appreciated as described below, that a variety of inventiveconjugates can be synthesized according to the methods described herein.The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCompany (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis,Mo.), or are prepared by methods well known to a person of ordinaryskill in the art following procedures described in such references asFieser and Fieser 1991, “Reagents for Organic Synthesis”, vols 1-17,John Wiley and Sons, New York, N.Y., 1991; Rodd 1989 “Chemistry ofCarbon Compounds”, vols. 1-5 and supps, Elsevier Science Publishers,1989; “Organic Reactions”, vols 1-40, John Wiley and Sons, New York,N.Y., 1991; March 2001, “Advanced Organic Chemistry”, 5th ed. John Wileyand Sons, New York, N.Y.; Larock 1990, “Comprehensive OrganicTransformations: A Guide to Functional Group Preparations”, 2^(nd) ed.VCH Publishers; and other references more specifically drawn to polymerchemistry. The methods described below are merely illustrative of somemethods by which the polyal conjugates of this invention can besynthesized, and various modifications to these methods can be made andwill be suggested to a person of ordinary skill in the art having regardto this disclosure.

The starting materials, intermediates, and conjugates of this inventionmay be isolated and purified using conventional techniques, includingfiltration, distillation, crystallization, chromatography, and the like.They may be characterized using conventional methods, including physicalconstants and spectral data.

Materials

Bovine pancreatic trypsin (EC 3.4.21.4) Type III, chymotrypsin,Nα-benzoyl-L-arginine ethyl ester (BAEE), acetyltyrosine ethyl ester(ATEE), dextran B-512 (Mn 188,000 Da) were obtained from Sigma ChemicalCompany (St Louis, Mo.). Sodium borohydride, sodium cyanoborohydride,sodium metaperiodate, 1-[3-(dimethylamino)propyl-3-ethylcarbodiimidehydrochloride (EDC), diethylenetriaminepentacetic acid (DTPA),4-dimethylaminopyridine (DMAP) and succinic anhydride were from Aldrich,St Louis, Mo. InCl₃ [In-111] was from Perkin Elmer Life Sciences(Boston, Mass.). Anhydrous pyridine, ethyl alcohol, and other solventswere obtained from Sigma-Aldrich and used without further purification.

Equipment and Methods

Size exclusion chromatography in aqueous media was carried out usingVarian-Prostar HPLC system equipped with BIO-RAD model 1755 RefractiveIndex detector and LDC/Milton Roy SpectoMonitor 3000 UV detector. HPSECcolumns, Biosil SEC-125 and Biosil SEC-400 (BIO-RAD), and low pressureSuperose-6 column (Pharmacia), were used for studying MW/MWD of polymersand polymer-protein conjugates. SEC column calibration was performedusing protein standards and broad molecular weight dextran standards.Unless otherwise stated, elution was performed isocratically in 50 mMpH=7.0 phosphate buffer with 0.9% NaCl. ¹H and ¹³C NMR were carried outon Varian Mercury-300, Bruker DPX-300, and Bruker Aspect 3000 NMRspectrometers using solvent peak as reference standard. Cary 300BioUV/visible spectrophotometer equipped with Peltier-thermostatedmulti-cell block was used for spectroscopic measurements and enzymekinetics studies. Radioactivity measurements were carried out usingWallac Wizard 1480 gamma counter (Perkin Elmer). Gamma scintigraphy wasperformed using Ohio Nuclear gamma camera with medium energy collimator.

Example 1 Exemplary Synthesis of Bifunctional Compounds of the Invention(Scheme 1)

General Methods. ¹H NMR spectra were recorded with a Varian XL-500spectrometer. Chemical shifts are expressed in parts per million (ppm)on the δ scale relative to a TMS internal reference standard. Ingeneral, CDCl₃ was used for the free bases and DMSO-d₆ was used forsalts. Coupling constants (J values) were given in Hz. Thin layerchromatography (TLC) was performed on 250 μm thickness silica gel platesor alumina precoated plates (Whatman, AL SIL G/UV or J. T. Baker,Baker-flex, SILICA GEL IB-F) containing fluorescent indicator (2×8 cm).Column chromatography was performed on silica gel (Baker, 40 μm Flashchromatography). Fractions were analyzed using TLC and compounds werevisualized using ninhydrin (0.5 g in 100 mL of methanol) for primary andsecondary amine(s), ultraviolet light and/or iodine vapor.

8-[1-Ethoxyethylideneaminooxy]octanoic acid 2,5-dioxo-pyrrolidin-1-ylester (6). To a mixture of ethyl N-hydroxyacetimidate 1 (6.2 g, 60 mmol)and 8-bromooctanoic acid (15 g, 67 mmol) in 150 mL of solvent mixture(methanol 8: acetone 1) was added drop wise 22 mL of 10N NaOH and heatedat 50° C. for 12 h. The reaction mixture was cooled, the organic solventwas removed by rotary evaporation. The dilute HCl solution was added toadjust pH down to 6. The resulting turbid mixture was extracted withCHCl₃ and EtOAc. The extract was washed with brine, dried, filtered, andsolvent was evaporated to crude oil (3, 12 g, 49 mmol, 82%). A mixtureof 3 (12 g, 49 mmol) and N-hydroxysuccinimide (5 g, 50 mmol) in 100 mLof THF was treated with 50 mL of DCC (1.0 M solution in CH₂Cl₂). Thesolids were filtered, and the clear filtrate was evaporated. Theresultant brown oil was purified on a silica gel column. Elution with20% ethyl acetate/hexane afforded 5.8 g (17 mmol, 34%) of the product asa wet solid. ¹H NMR (DMSO-d₆): δ 1.21 (3H, t, J=7.0), 1.28-1.38 (6H, m),1.57 (2H, p, J=6.8), 1.64 (2H, p, J=7.4), 1.86 (3H, s), 2.65 (2H, t,J=7.5), 2.81 (4H, s), 3.82 (2H, t, J=6.5), 3.94 (2H, q, J=7.0)

8-Aminooxy-octanoic acid 2,5-dioxo-pyrrolidin-1-yl ester HCl salt (10).To a solution of 6 (5.8 g, 17 mmol) in THF (30 mL) was added a mixtureof HCl and THF (4 mL, 2 mL of c.HCl and 2 mL of THF. The reactionmixture was stirred for 4 h, concentrated to a wet solid and washed with5% EtOAc/hexane. The product solidified (hygroscopic) under vacuum wasused without further purification. ¹H NMR (DMSO-d₆): δ 1.28-1.37 (6H,m), 1.57-1.65 (4H, m), 2.65 (2H, t, J=7.3), 2.82 (4H, s), 4.02 (2H, t,J=6.3), 11.09 (3H, br s).

Example 2 Synthesis of Maleimidyl Bifunctional Linkers for PolymerConjugates

Using the same protecting group, aminooxy alkyl maleimide wassynthesized as shown in Scheme 2.

3-[5-Carboxypentylcarbamoyl]acrylic acid (14). 6-Aminocaproic acid (10.1g, 81.6 mmol) was added to maleic anhydride (8.09 g, 82.6 mmol), in 100mL of anhydrous DMF. The mixture was stirred at room temperature for 24h and poured into 250 mL of water. White precipitate was filtered,washed with ether and dried (15 g, 86%). ¹H NMR (DMSO-d₆): δ 1.27-1.33(2H, m), 1.45-1.54 (4H, m), 2.21 (2H, t, J=7.3), 3.18 (2H, q, J=6.7),6.24 (1H, d, J=13.0), 6.42 (1H, d, J=12.5), 9.11 (1H, br s), 13.6 (1H,br).

6-[2,5-Dioxo-2,5-dihydro-pyrrol-1-yl]hexanoic acid (15). The acid 14(4.5 g, 21 mmol) was refluxed in 60 mL of (Ac)₂O with NaOAc (1.72 g, 21mmol) for 3 h. After cooling, the reaction mixture was concentrated. Theresidue was dissolved in EtOAc, washed with brine, dried over MgSO₄,filtered and evaporated to give a deep red oil that was purified bysilica gel column chromatography (CHCl₃:MeOH/99:1). The maleimideproduct 15 was isolated as a white solid (1.4 g, 32%). ¹H NMR (DMSO-d₆):δ 1.18-1.24 (2H, m), 1.45-1.52 (4H, m), 2.18 (2H, t, J=7.5), 3.38 (2H,t, J=7.0), 7.01 (2H, s).

N-{3-[6-[2,5-Dioxo-2,5-dihydro-pyrrol-1-yl]-hexanoylamino]-2-hydroxy-propyl}-acetimidicacid ethyl ester (16). The amine 13 (1.6 g, 10 mmol), in 30 mL ofanhydrous THF was added dropwise to a solution of 10 mL of DCC (10 mmol,1M solution of DCC in CH₂Cl₂) and acid 15 (2.1 g, 10 mmol). The mixturewas stirred at room temperature for 24 h. The reaction mixture wasconcentrated. The residue was dissolved in EtOAc, washed with brine,dried over MgSO₄, filtered and evaporated to give a colorless oil thatwas purified by silica gel column chromatography (CHCl₃:MeOH/99:1). Theamide product 16 was isolated as a wet solid (1.5 g, 42%). ¹H NMR(CDCl₃): δ 1.27 (3H, t, J=7.3), 1.30-1.35 (2H, m), 1.60 (2H, p, J=7.3),1.67 (2H, p, J=7.8), 1.94 (3H, s), 2.20 (2H, t, J=7.5), 3.20-3.25 (1H,m), 3.51 (2H, t, J=7.3), 3.48-3.55 (1H, m), 3.87 (1H, dd, J=6.3, 11.8),3.92-4.01 (5H, m), 4.04 (1H, br s), 6.34 (1H, t, J=5.5), 6.71 (2H, s).

6-[2,5-Dioxo-2,5-dihydro-pyrrol-1-yl]-hexanoic acid[3-aminooxy-2-hydroxy-propyl]-amide HCl salt (17) was prepared asdescribed for 10 to give the title compound 17 as a white solid.

REFERENCES

-   (1) Stanek et. al. J. Med. Chem. 1992, 35, 1339-1344.-   (2) Buehler et. Al. J. Am. Chem. Soc. 1967, 89, 261-265

Example 3 Biodegradable Hydrophilic Polyals for Protein Modification

As discussed above, biodegradation of macromolecular therapeutics is animportant but incompletely studied issue, even for most widely usedpolymers. For example, there is a potential risk that extended clinicaluse of conjugates containing non- or slow-biodegradable polymerfragments can lead to long-term cell vacuolization (see, for example,Bendele A. Seely J. Richey C. Sennello G. Shopp G. (1998) Shortcommunication: renal tubular vacuolation in animals treated withpolyethylene-glycol-conjugated proteins. Toxicological Sciences. 42,152-7) and overload, development of lysosomal disease syndrome (see, forexample, Christensen, M., Johansen, P., Hau C., (1978) Storage ofpolyvinylpirrollidone (PVP) in tissue following long-term treatment witha PVP-containing Vasopressin preparation. Acta Med Scand., 204,295-298), and, at higher doses, to other pathological metabolicalterations (see, for example, Miyasaki K. (1975) Experimental PolymerStorage Disease in Rabbits. Virchows Arch. A. Path. Anat. And Histol.,365, 351-365). The predominant clearance route of relatively large(>10-15 nm) long circulating conjugates, regardless of the size of thepolymer component, is through uptake by cells (mostly in RES, but alsoin other tissues) followed by intracellular degradation andmetabolization. Reducing the molecular weight of the polymer component,e.g. to 30-40 kDa, which is an effective strategy for enabling renalclearance of small molecule drug conjugates (see, for example, Duncan,R., Gac-Breton, S., Keane, R., Musila, R., Sat, Y. N., Satchi, R.,Searle, F. (2001) Polymer-drug conjugates, PDEPT and PELT: basicprinciples for design and transfer from laboratory to clinic, J.Controlled Release, 74, 135-146), is not a feasible solution for proteinconjugates or other large (>5-7 nm) constructs. Conjugates degradingupon cell uptake with release of smaller but still non-biodegradablefragments, such as PEG telomers with degradable linkages between PEGblocks (See, for example, Tomlinson R, Klee M, Garrett S, Heller J,Duncan R and Brocchini S. (2002) Pendent Chain FunctionalizedPolyacetals That Display pH-Dependent Degradation: A Platform for theDevelopment of Novel Polymer Therapeutics, Macromolecules, 35, 473-480),would most unlikely not solve the problem because no efficient cellularmechanisms transporting such fragments back to the extracellular spacehave been identified. Development of essentially completelybiodegradable polymers, preferably degrading with formation oflow-toxicity, readily clearable or metabolizable products, appear to bethe predominant possible radical solution of the problem of long-termintracellular deposition. The type of protein-polymer linkage and thedegree of polymer modification can also alter both conjugatedegradability and biological properties (see, for example,Danauser-Reidl, S., Hausmann, E., Schinck, H., Bender, R.,Dietzfilbinger, H., Rastetter, J., Hanauske, A. (1993) Phase-I clinicaland pharmacokinetic trial of Dextran conjugated Doxorubicin (AD-70,DOX-OXD). Invest. New Drugs, 11, 187-195). This necessitates theselection of a polymer backbone structure and conjugation strategiesthat would not interfere, or minimally interfere, with biologicalfunctions of the protein component, nor (where applicable) adverselyalter protein properties upon release from the conjugate. A combinationof a macromolecular material and a cross-linking reagent enablingsufficient conjugate stability in the normal extracellular environmentand, on the other hand, acceptable rate of conjugate disintegration uponendocytosis, would be most beneficial.

Hydrophilic essentially fully degradable polyals, e.g.,poly[1-hydroxymethylethylene hydroxymethyl-formal] (PHF), have beendeveloped and reported as acyclic mimetics of polysaccharides (see, forexample, (1) Papisov M I, Garrido L, Poss K, Wright C, Weissleder R,Brady T J. (1996) A long-circulating polymer with hydrolizable mainchain. 23-rd International Symposium on Controlled Release of BioactiveMaterials, Kyoto, Japan, 1996; Controlled Release Society, Deerfield,Ill.; 107-108; and (2) Papisov M. I. (1998) Theoretical considerationsof RES-avoiding liposomes. Adv. Drug Delivery Rev., 32, 119-138). Thesematerials, which can be prepared synthetically and by lateral cleavageof some polysaccharides, were shown to be essentially (i)non-bioreactive, (ii) non-toxic and (iii) fully degradable, and, thus,proved to have potential in various pharmaceutical applications (see,for example, (1) Papisov M I, Babich J W, Dotto P, Barzana M, Hillier S,Graham-Coco W, Fischman A J. (1998) Model cooperative (multivalent)vectors for drug targeting. 25th Int. Symp. on Controlled Release ofBioactive Materials, 1998, Las Vegas, Nev., USA; Controlled ReleaseSociety, Deerfield, Ill., 170-171; and (2) Papisov M I. (2001) Acyclicpolyacetals from polysaccharides. (Biopolymers from polysaccharides andagroproteins), ACS Symposium Series 786, pp. 301-314). Polyals containpH-sensitive acetal or ketal groups within the main chain, whichprovides the desired combination of polymer stability in neutral andalkaline media and destabilization in acidic environment.

In certain embodiments, the present invention further expands the scopeof potential applications for hydrophilic polyals, and demonstratessuitability of these materials for preparation of essentially fullydegradable protein conjugates with preservation of proteinfunctionality. In certain exemplary embodiments, a hydrophilic polyal(PHF) is used to obtain and characterize conjugates of well-known modelproteases, trypsin and α-chymotrypsin. Conjugation techniques includethe use of new bifunctional coupling reagents containing an aminooxy(O-hydroxylamino) group; these reagents were also developed in ourlaboratory and specifically tailored for conjugations involvingaldehyde-bearing molecular modules in aqueous media. The main modelprotein of this study, trypsin, was selected as a relatively smallprotein with readily measurable activity and fast blood clearance.Trypsin was also well characterized in immobilization reactionsinvolving various soluble and solid carriers and conjugation techniques.

Experimental Section

Materials

Bovine pancreatic trypsin (EC 3.4.21.4) Type III, chymotrypsin,N□-benzoyl-L-arginine ethyl ester (BAEE), acetyltyrosine ethyl ester(ATEE), dextran B-512 (Mn 188,000 Da) were obtained from Sigma ChemicalCompany (St Louis, Mo.). Sodium borohydride, sodium cyanoborohydride,sodium metaperiodate, 1-[3-(dimethylamino)propyl-3-ethylcarbodiimidehydrochloride (EDC), diethylenetriaminepentacetic acid (DTPA),4-dimethylaminopyridine (DMAP) and succinic anhydride were from Aldrich,St Louis, Mo. InCl₃ [In-111] was from Perkin Elmer Life Sciences(Boston, Mass.). Anhydrous pyridine, ethyl alcohol, and other solventswere obtained from Sigma-Aldrich and used without further purification.

Equipment and Methods

Size exclusion chromatography in aqueous media was carried out usingVarian-Prostar HPLC system equipped with BIO-RAD model 1755 RefractiveIndex detector and LDC/Milton Roy SpectoMonitor 3000 UV detector. HPSECcolumns, Biosil SEC-125 and Biosil SEC-400 (BIO-RAD), and low pressureSuperose-6 column (Pharmacia), were used for studying MW/MWD of polymersand polymer-protein conjugates. SEC column calibration was performedusing protein standards and broad molecular weight dextran standards.Unless otherwise stated, elution was performed isocratically in 50 mMpH=7.0 phosphate buffer with 0.9% NaCl. ¹H and ¹³C NMR were carried outon Varian Mercury-300, Bruker DPX-300, and Bruker Aspect 3000 NMRspectrometers using solvent peak as reference standard. Cary 300BioUV/visible spectrophotometer equipped with Peltier-thermostatedmulti-cell block was used for spectroscopic measurements and enzymekinetics studies. Radioactivity measurements were carried out usingWallac Wizard 1480 gamma counter (Perkin Elmer). Gamma scintigraphy wasperformed using Ohio Nuclear gamma camera with medium energy collimator.

Polymer Synthesis

PHF is a semi-synthetic acyclic polyacetal which can be prepared vialateral cleavage of Dextran B-512 with periodate. Dextran B512, aproduct of Leuconostoc Mesenteroides strain B-512, is a nearly linear(1->6)-poly-α-D-glucose with ca. 5% (1→3; β) branching, of which 95% areonly one or two residues long (see, for example, Jeanes A. (1986)Immunochemical and related interactions with dextrans reviewed in termsof improved structural information. Molecular Immunology 23, 999-1028).Periodate oxidation of (1->6)-polyglycoside in controlled conditionsstarts with breaking up ether C2-C3 or C3-C4 bond, resulting in theformation of dialdehydes IIa and IIb (see, for example, Ishak M. F.,Painter T. J., (1978) Kinetic evidence for hemiacetal formation duringthe oxidation of dextran in aqueous periodate. Carbohyd. Res., 64,189-97). The slower oxidation stage, cleavage of C3, leads to dialdehydeIII (Scheme 3).

Borohydride reduction of aldehyde groups of dialdehydes IIa, IIb and IIIgives polyals with pendant hydroxymethyl groups IV and (from IIa andIIb) vicinal glycol groups V. Accurate control of oxidizer/substratestoichiometry and reaction conditions enables generation of polymerswith a desirable amount of vicinal diol, which can be subsequently usedas selective reactive sites for further polymer modification andconjugation. In certain embodiments, both PHF and PHF-diols with vicinaldiol content ranging from 2 to 20% (mol) were prepared and used asprotein carriers. In most cases, dextran with number average molecularweight (Mn) of 188 kDa was used as starting material.

Poly-1-hydroxymethylethylene hydroxymethyl-formal, IV (PHF). In certainembodiments, PHF was prepared via exhaustive lateral cleavage ofcarbohydrate rings by periodate oxidation. Dextran of Mn 188,000 Da(15.15 g, 93.4 mmol of glycopyranoside) was dissolved in 300 ml ofdeionized water. The Dextran solution was treated with 47.95 g (224.2mmol) of metaperiodate dissolved in 350 ml of deionized water at 0-5° C.in a light protected reactor for 3 hours. The precipitated sodium iodatewas removed by filtering the reaction mixture through 1μ glass filter.The pH of the filtrate was adjusted to 8.0 with 5N NaOH, and theresultant solution was treated with sodium borohydride (7.4 g, 200 mmol,dissolved in 100 ml of deionized water) for 2 hours. Then, the pH of thereaction medium was adjusted to approximately 6.5 with 1 N HCl. Theobtained macromolecular product was purified and concentrated on CH2PRflow dialysis system (Amicon, Beverly, Mass.) equipped with hollow fibercartridge, cutoff 30 kDa, by passing approximately 4 volumes ofdeionized water through the polymer solution. Alternatively, the productwas purified on G-25 preparative column using deionized water as aneluent. PHF was recovered from aqueous solutions by lyophilization.Average polymer yields ranged from 70% to 80%. SEC analysis of a typicalPHF prepared from Dextran 188 kDa showed peak molecular weight at130,000 Da, Mn 92,000 Da, and polydispersity index (Mw/Mn) of 2.5. Thestructures of all obtained polymers, as examined by ¹³C and ¹H NMR, wereconsistent with the expected acyclic polyacetal structure. The typicalsynthetic procedures for PHF and PHF-glycol preparation described below.

PHF-glycol. In certain embodiments, PHF-glycol was prepared bycontrolled dextran cleavage that was stopped at stage II. Polymerscomprising vicinal diol structural units were obtained as a result ofsubsequent reduction from intermediates IIa and IIb. Glycol-substitutedpolymers were prepared as described above for PHF, except that thestarting (glucopyranoside)/(periodate) molar ratio was 1.00 to 0.95. Thepresence of PHF-diol structure VI in the resultant polymer was confirmedby ¹H NMR spectroscopy. Polymer spectrum registered in DMSO-d₆:D₂O (95:5v/v) has shown the specific for structure IV signal of C1-H at δ 4.62(t, J=5.2 Hz) and the signal of C1 acetal hydrogen at δ 4.49 (d, J=5.2Hz) characteristic for structure V. At the same time, no C4-H signals atδ 3.10-3.20 (m) were registered, indicating the absence of C₃-C₄ diolsin reduced IIb. The amount of PHF-diol structures (V), as determined byNMR, was approximately 2%. SEC analysis has shown no substantialdifference between MW/MWDs of the starting Dextran and the resultantPHF-glycol.

PHF succinate (PHF-SA). PHF (100 mg), succinic anhydride (7.5 mg, 0.075mmol) and DMAP (1.2 mg, 0.01 mmol) were dissolved in 5 ml of anhydrouspyridine. After 18 hours of agitation at 40° C., pyridine was removed invacuum. The residue was suspended in deionized water, and the pH wasadjusted to 7.0 by addition of 1 N NaOH. The succinylated PHF waspurified on a Sephadex G-25 column with deionized water an eluent, andrecovered from aqueous solution via lyophilization. The succinic acidcontent, as determined by potentiometric titration, was 11.3%. The ¹HNMR spectrum of the polymer (D₂O) contained signals of characteristicmethylene protons of succinic acid ester at δ 2.62 (t) and δ 2.46 (t).

Protein Conjugates

In certain embodiments, Protein conjugates with PHF-SA were prepared viaEDC mediated coupling reaction PHF-SA with model proteins.

PHF-diol conjugates were prepared by conventional reductive amination(see, for example, Dottavio-Martin, D. and Ravel, J. M., (1978)Radiolabeling of proteins by reductive alkylation with[14C]-formaldehyde and sodium cyanoborohydride. Analyt. Biochem., 87,562) of polymeric aldehydes generated from vicinal glycol groups presentin PHF-diol, as well as by non-reductive amination utilizing theaminooxy (O-hydroxylamino)-containing bifunctional agents of theinvention.

Two types of aminooxy-reagents were developed (see examples 1 and 2above) and tested, containing a protected aminooxy-group and either amaleimide group for thiol modification(N-(5-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-5-oxo-hexenyloxy)-acetimidicacid ethyl ester, VII), or N-hydroxysuccinimide ester group foraminogroup modification(N-(3-(3-(2,5-dioxo-pyrrol-1-yl)-propionylamino)-2-hydroxy-propoxy)-acetimidicacid ethyl ether, VIII). Non-reductive coupling of the aminooxy couplingreagents, or their unprotected analogs, with carbonyl containingcompounds was conducted at pH 3-4, which enabled preservation ofN-oxysuccinimido- and maleimido-functions for the subsequent stage ofprotein coupling. Representative procedures illustrating the process ofPHF conjugation are given below for trypsin.

PHF-trypsin conjugates (reductive amination). The solution of PHF-diolwith Mn 150 kDa (200 mg) and diol content 10% (mol/mol monomer) wasdissolved in 2 ml of deionized water and combined on ice with 30.1 mg(0.14 mmol) of NaIO₄ in 0.25 ml of deionized water. After a 1 hourincubation, the activated polymer was combined with 31.8 mg of trypsinin 6.0 ml of 0.1 M phosphate buffer pH 5.5 and 18 mg (0.29 mmol) sodiumcyanoborohydride, incubated on ice for 1 hour, then at 8° C. for 18hours. The macromolecular product was recovered by gel filtration onSephadex G25 column equilibrated with deionized water, and separatedfrom unreacted trypsin on Superose-6 column. Trypsin conversion was 61%(HPSEC BioSil-125, detected by UV at 280 m).

PHF-SA-trypsin conjugates. PHF-SA solution with Mn=176 kDa, 100 mg in2.0 ml of deionized water, was combined with 3.0 ml of 5.0 mg/ml trypsinsolution in 0.1M phosphate buffer, pH 7.4. Then, EDC (20 mg) was addedto the reaction mixture in 500 μl of cold (0-5° C.) deionized water.Trypsin conversion after 3 hours of incubation, according to HPLC (UV at280 nm), was 97%. The reaction mixture was separated from low molecularweight components and concentrated to approximately 10 mg/ml on PM-30ultrafiltration membrane using 0.05 M PBS pH 7.0. The conjugate wasseparated from residual unbound trypsin on Superose-6 column with 0.5MPBS, pH 7.0, as a running buffer. The resultant conjugate was aliquotedand stored frozen at −40° C. SEC analysis of this conjugate gave Mn=245kDa, PI=1.8, and peak polymer MW=260 kDa. Trypsin conjugate contentestimated by HPLC and spectroscopically at 280 nm was 10.7% w/w.

PHF-AO(NHS)-trypsin conjugates. The solution of PHF-diol with Mn ˜150kDa (200 mg) and diol content 2% (mol/mol monomer) was dissolved in 2 mlof deionized water and combined on ice with 15 mg (0.07 mmol) of NaIO₄in 0.125 ml deionized water. After a 1 hour incubation, the polymerproduct was purified by gel filtration on Sephadex G-25, using deionizedwater as an eluent. The resulting solution was diluted with 3.0 ml ofethyl alcohol and combined with 92.6 mg of VIII. in 2 ml of ethanol. ThepH of the mixture was adjusted to 3.0 by addition of 1M NaHSO₄, andagitated for 2 hours on ice. The pH was adjusted to approximately 7.0,and the product was purified on a Sephadex G-25 column equilibrated withdeionized water. The resulting product was combined with 32 mg oftrypsin dissolved in phosphate buffer pH 8.6, and the mixture wasincubated on ice for 3 hours. HPSEC (UV at 280 nm) analysis of thereaction mixture showed 98% trypsin bonding to the polymer. The reactionmixture was desalted and concentrated to approximately 10 mg/ml on PM-30ultrafiltration membrane using 0.05 M PBS pH 7.0. The conjugate wasseparated of residual unbound trypsin on Superose-6 column (Pharmacia)with 0.5M PBS pH 7.0 as a running buffer. The resultant conjugate wasaliquoted and stored frozen at −40° C.

PHF-AO(MI)-trypsin conjugates. PHF-aldehyde was prepared as describedabove from PHF diol, Mn ˜150 kDa. The PHF diol, 100 mg in 5.0 ml ofdeionized water, was combined with 46 mg of VII. The pH of the mixturewas adjusted to 3.0 by addition of 1M NaHSO₄ and agitated for 2 hours onice. Then, the pH was adjusted to 6.5, and the polymer was desalted onSephadex G-25 column equilibrated with deionized water. The obtainedproduct was combined with 16 mg of trypsin dissolved in phosphatebuffer, pH 8.6, and incubated for 2 hours on ice and for 18 hours at 8°C. HPSEC (UV at 280 nm) analysis of the reaction mixture showed 75%trypsin bonding to polymer carrier. The reaction mixture was desaltedand concentrated to approximately 10 mg/ml on a PM-30 ultrafiltrationmembrane, using 0.05 M PBS, pH 7.0. The conjugate was separated from theresidual unbound trypsin on Superose-6 column with 0.5M PBS, pH 7.0, asa running buffer, the desalted on Sephadex G-25 column equilibrated withdeionized water, and lyophilized. SEC analysis of this conjugate (Biosil400) showed substantial presence of a high molecular weight fractioneluted with void volume. Peak MW ˜500 kDa.

Trypsin Conjugate Modification with DTPA and ¹¹¹In Labeling.

For animal studies, protein conjugates were labeled with [¹¹¹In] aftermodification of the trypsin portion of conjugates with DTPA. EDCmediated coupling was carried out in aqueous solution atDTPA/EDC/Trypsin lysine residue ratio 500:50:1 at pH 7.5. The resultantDTPA-labeled conjugates were purified by gel chromatography on SephadexG-25. DTPA to protein molar ratio, as determined by Cu(II) colorimetricassay at 775 nm was approximately 1:4. Unmodified proteins Were labeledanalogously.

Labeling was performed by transchelation from [¹¹¹In] citrate. Thelabeling solution was prepared by mixing carrier-free [¹¹¹In] indiumchloride in 0.05 M HCl with a 20-fold volume excess of 0.5 M sodiumcitrate, pH=5.6. The resultant [¹¹¹In] indium citrate solution was addedto unbuffered solutions of DTP-modified polymers at 0.2 to 1 mCi of[¹¹¹In] per 1 mg of dry substance. The labeled conjugates were separatedby gel chromatography on Sephadex G-25, with simultaneous mediareplacement to sterile isotonic saline. Labeling efficacy aftertranschelation, as estimated by HPLC equipped with gamma detector, onaverage exceeded 90%. Radiochemical purity after desalting was >99%.

Blood Clearance and Biodistribution Study

Animal experiments were performed in accordance with institutionalguidelines. Adult male CD1 mice (weight in a range 28 g to 34 g, CharlesRiver Laboratories, Wilmington, Mass.) were injected with labeledconjugates and unmodified proteins via the tail vein (150 μL perinjection, containing approximately 10 μCi of ¹¹¹In).

Mice were euthanized at 0.25, 0.5, 1, 2, 4, and 8 hours (n=2). Bloodsamples and harvested organs (hart, lungs, liver, spleen, kidneys,adrenal glands, stomach, GI, testes, muscle, bone, brain and tail) wereanalyzed on gamma counter. The amount of radioactivity per organ wasexpressed as a percentage of the injected dose per gram tissue. For thePHF-AO-Trypsin (2:1) conjugate, blood clearance data were adjusted forunbound trypsin content (15% mol) using unmodified trypsin clearanceprofile for background correction.

Enzyme Activity

Esterase activity of model enzymes was measured with BAEE (trypsin),ATEE (α-chymotrypsin), and BSA as substrates at pH 7.4, followingpublished techniques (see, for example, Foucault, G., Seydoux, F., Yon,J. (1974) Comparative kinetic properties of alpha, beta and psi form oftrypsin. Eur. J. Biochem. 47, 295-302).

Hydrolytic Stability of Conjugates

Hydrolytic degradation of PHF and PHF-trypsin conjugates was studied at37° C. in PBS at pH 7.4 and 5.5. The pH of the media remained constantover the course of the experiment. HPSEC analysis (Biosil 400) of thereaction mixture aliquots taken at 24, 72 and 144 hours was carried outto monitor the MW/MWD and composition of degradation products. Thecontent of unbound and PHF-associated trypsin were estimated bymonitoring absorbance at 280 nm.

PHF Conjugation with Proteins.

In certain embodiments, model proteins, trypsin and α-chymotrypsin, wereused as models for development and characterization of protein-polyalconjugates. Conjugates were prepared utilizing three differentcrosslinking approaches.

The first approach was based on acylation of primary alcoholfunctionality present in PHF with succinic anhydride. Proteinconjugation was subsequently conducted via by carbodiimide mediatedcoupling of the carboxy-modified polymer with the model protein (themethod is targeted to coupling through, predominantly, lysine moieties).In the conditions used, approximately four of eight lysine moieties pertrypsin and 14 moieties per chymotrypsin molecule were expected to bereactive. This approach was found to be productive, giving up to 95-98%protein conjugation and 90-95% preservation of protein activity in theconjugates.

Another conjugation technique used herein was based on activation ofpendant glycol groups introduced into PHF structure via reduction of theintermediate oxidation products IIa/IIb (Scheme 3). Diols are readilytransformed into active aldehyde groups immediately prior toconjugation. In one example, PHF-diol polyal protein conjugate wasprepared by conventional method of reductive amination of polymericaldehydes (conjugate 1, Table 1). Protein conversion, even at highaldehyde content in the polymer, was relatively low (−60%).

TABLE 1 Composition and MW/MWD of PHF-protein conjugates Protein Enzymeto con- PHF ratio version, # Carrier Crosslinker Enzyme (mol) % 1PHF-diol Direct Trypsin 1 61 amination 2 PHF SA Trypsin 1 97 3 PHF-diolAO-NHS (VIII) Trypsin 1 98 4 PHF-diol AO-NHS (VIII) Trypsin 2 85 5PHF-diol AO-NHS (VIII) Trypsin 4 72 6 PHF-AO- N/a Trypsin 1 87 NHS* 7PHF-diol AO-MI (VII) Trypsin 1 75 8 PHF-diol SA Chymotrypsin 1 95 9PHF-diol AO Chymotrypsin 1 93 *PHF-glycol with 2,3-diol content 2% wasmodified with VIII. Isolated and lyophilized PHF-AO-NHS was usedsequentially for preparation of protein conjugate in one step withoutadditional activation.

Alternatively, conjugates 2-7 and 9 were prepared utilizing theinventive aminooxy-group containing bifunctional reagents VII and VIIIdeveloped for conjugation of carbonyl containing compounds withmolecules containing amino and sulfhydryl groups, respectively (Scheme4).

The method was developed as a replacement for the widely usednon-reductive coupling of aldehydes and hydrazones. Amination withaminooxy-reagents requires milder conditions, e.g., at pH as high as 6if necessary, and results in a more stable oxime bond (the stability ofhydrazone conjugates are limited even at pH 7-7.5 [ see, for example,Shan. S. Wong. Chemistry of protein conjugation and cross-linking. CRCPress, 1993]).

Scheme 4. Conjugation of Proteins with PHF Using Aminooxy-NHS andAminooxy-Maleimide Crosslinkers.

Application of aminooxy compounds for fast, one-step coupling throughcarbonyl groups (ketones, aldehydes) with formation of oxime bonds iswidely used in medicinal chemistry and, in bioconjugate chemistry, wasdescribed, for example, for coupling glycosylated proteins with amino(see, for example, Berninger, R. W. Aminooxy-containing linker compoundsand their application in conjugates. PCT WO 96/40662) and sulfhydrylgroup containing ligands (see, for example, Webb, R. R., II, and Kancko,E. (1990) Synthesis of 1-(aminooxy)-4-[(3-nitro-2-pyridyl)dithio]butanehydrochloride and of1-(aminooxy)-4-[(3-nitro-2-pyridyl)dithio]but-2-ene. Novelheterofunctional cross-linking reagents, Bioconjugate chem., 1, 96). Thebifunctional reagents of the invention contain a free or protectedaminooxy-group and either a thiol reactive maleimide group (VII) or anamino reactive N-hydroxysuccinimide ester group (VIII). Non-reductivecoupling of these aminooxy coupling reagents with aldehydes wasconducted in water at pH 3 for 2 hours. When unprotected analogs of VIIand VIII or their hydrochloride salts were used, the pH of the reactionmixture during the coupling was maintained in the range of 3.5-4.5. Mildacidic conditions in both cases enabled preservation ofN-oxysuccinimido- and maleimido-functions for the subsequent stage ofprotein coupling. Glycol content in PHF-diol as low as 2% mol. (orapproximately 20 diol moieties per PHF molecule of MW=150 kDa) wassufficient to achieve quantitative coupling of proteins at PHF toprotein 1:1 mol/mol.

Conjugates with molar protein to polymer ratio in the range from 1:1 to4:1 were successfully prepared utilizing the above mentioned strategies;in most cases, the desirable degree of modification was achieved withhigh yields (85-95%). Conjugates prepared in one step using aminooxy-NHScoupling reagent VIII and via EDC mediated coupling to PHF-SA gave thehighest conjugate yields (up to 95-98% for 1:1 conjugates) with respectto protein precursor. Both aminooxy reagents have shown high degree offlexibility with respect to the conjugation sequence and conditions.Different activation conditions, and sufficient stability of aminooxy,NHS and maleimido coupling groups, allowed to change the reaction orderat will, or, if required, to work with isolated and purified aminooxycontaining proteins or polyal intermediates. HPSEC data indicated that,in spite of the acidic conditions at the polyal/aminooxy coupling step,protein adducts were obtained without substantial depolymerization ofPHF backbone and with nearly theoretical yields.

Without wishing to be bound to any particular theory, we proposed thatthe observed conjugate MW (in many cases 1.5 to 2 times higher thanexpected) may be the result of partial crosslinking of polyal chains viaprotein modifier. This process, however, had no noticeable effects onenzymatic activity of conjugates.

Comparative evaluation of enzymatic activity was performed usingconjugates with approximately 1:1 carrier/protein molar ratio(conjugates 2, 3, and 8, Table 1). No significant changes in theMichaelis-Menten parameters (K_(M), k_(kat)) and no pH optimum shiftswere observed, as compared to native enzymes. Conjugates retained from85% to 95% of the native enzyme activity when tested with syntheticsubstrates. As expected, a somewhat more significant decrease in enzymeactivity was observed using BSA as a substrate, most likely due toexpected steric hindrance. Thus, the obtained data are in agreement withearlier literature data on trypsin conjugates, e.g., with partiallyoxidized sucrose polymers (see, for example, R. Vankatesh and P. V.Sundaram. (1998) Modulation of stability properties of bovine trypsinafter in vitro structural changes with a variety of chemical modifiers.Protein Engineering, 11, 8, 691-698).

-   -   pH-Dependent Hydrolytic Degradation of PHF-protein conjugates.

All PHF and PHF-diol based conjugates exhibited a pH-dependent profileof hydrolytic degradation, being essentially stable at neutral andslightly basic pH (7.0-10.5).

Hydrolytic degradation of PHF and two different PHF-trypsin conjugates,PHF-SA-Trypsin (protein content 10%, Mn=250 kDa) and PHF-AO-Trypsin(protein content 25%, Mn 350 kDa), was studied at pH 7.4 and 5.5.Indeed, neither substantial changes in polymer MW/MWD nor noticeableaccumulation of unbound trypsin were observed at 37° C. pH 7.4 over a144-hour period. On the contrary, incubation of polyals at 37° C., pH5.5, for the same time period, showed steady and slow decrease in Mn andbroadening of molecular weight distribution for all three preparations,and protein release for both conjugates (see Table 2). The data on i)the rate of Mn decrease, ii) the amount of trypsin released, and iii)the percentage of polymer fraction recovery, suggests that the oximelinked conjugate (PHF-AO-Trypsin) has a higher degradation rate then PHFand the succinic acid cross-linked PHF conjugate (PHF-SA-Trypsin).

TABLE 2 Degradation of PHF and PHF-trypsin conjugates in PBS at pH 5.5Time, Polymer Trypsin h Mn Peak MW PI recovery, % release, % PHF 092,000 130,000 2.6 100.0 n/a 72 86,000 99,000 2.0 98.5 ″ 144 66,00097,000 3.6 89.2 ″ PHF-AO-Trypsin (1:2) 0 344,000 429,000 2.5 100.0 n/a72 270,000 363,000 2.8 96.1 5.4 144 174,000 255,000 4.6 89.8 10.5 PHF-SA-Trypsin (1:1) 0 246,000 260,000 1.8 100.0 n/a 72 177,000 209,0002.0 99.8 4.7 144 153,000 176,000 3.1 98.6 5.4

Biokinetics and biodistribution of [¹¹¹In] DTPA labeled modelPHF-protein conjugates. Biokinetics of PHF-SA-Trypsin, PHF-AO-Trypsin(protein content 10% and 25%, and Mn 250=kDa and 350 kDa respectively)and unmodified trypsin (26 kDa) were studied to determine the effect ofPHF modification on protein biokinetics and biodistribution. The datashowed significant improvements in blood half-life of PHF modifiedtrypsin, as compared to the unmodified protein (FIG. 1).

The unmodified radiolabeled trypsin preparation showed clearance of 80%of activity from blood within 15 minutes after administration (initialblood half life ca. 7 min), followed by an apparently monoexponentialclearance with 4±0.8 hour half-life. Considering the relatively smallprotein size and the character of final biodistribution, the first(main) phase is consistent with renal clearance and extravasation. Thesecondary phase can be related to redistribution back from the tissuesand prolong circulation of trypsin complexes with macromolecularprotease inhibitors present in plasma.

Both PHF-Trypsin conjugates also showed a biphasic blood clearancepattern, although of a different character and length. Afterapproximately 40% of activity was cleared from blood within 1 hour, therest (main fraction) remained in circulation with a half-life time of 8hours. Here, the first phase is consistent with extravasation (and,possibly, partial renal clearance) of the smaller fraction of theunfractionated conjugates, whereas the second phase is consistent withlong circulation of the main fraction of the conjugate. Notably, thelong circulation also indicates preservation of conjugate integritywithin the timeframe of the experiment.

The final biodistribution data (Table 3) showed an up to one order ofmagnitude reduction in label accumulation in kidneys, and a significantreduction in hepatic accumulation for polyal conjugates versusunmodified trypsin. No notable increase of accumulation in spleen (whichis sometimes observed for high molecular weight polymers andmicroparticles) was observed for PHF conjugates as compared to trypsincontrol.

Note that animals were not perfused, and therefore the higher labelcontent in lung tissue (both PHF-AO- and PHF-SA-Trypsin) and hart(PHF-AO-Trypsin) is consistent with higher residual conjugate content inthe blood pool of these organs.

TABLE 3 Biodistribution of radiolabeled conjugates at 8 hours followingiv administration Label accumulation, % dose/g tissue PHF-SA- PHF-AO-Tissue Trypsin Trypsin Trypsin Blood 7.4 13.0 2.2 Hart 1.1 2.8 0.8 Lung4.2 6.6 2.4 Liver 5.3 5.0 7.9 Spleen 2.5 2.9 2.1 Kidney 3.1 7.7 35.8Adrenals 1.2 2.5 1.3 Stomach 0.5 1.0 0.5 GI 0.7 1.0 0.8 Testes 0.4 0.60.8 Muscle 0.3 0.5 0.2 Bone 1.5 2.0 1.1 Brain 0.2 0.4 0.1

A detailed in this Example, model protein conjugates of PHF, ahydrophilic polyals, have been successfully prepared. Along with theconventional conjugation techniques employing acylation-basedcrosslinking, the bifunctional reagents of the invention allowing theformation of oxime bonds were used. Linkages formed by such reagentshave the same pH sensitivity profile as the PHF backbone. PHF is ahighly hydrophilic, essentially non-toxic semi-synthetic polymer (notoxicity in mice at 4 g/kg iv; See, for example, M Papisov et al.Semi-synthetic hydrophilic polyals. Under review (2002)) stable inphysiological conditions but undergoing non-enzymatic hydrolysis atlysosomal pH. All previously tested PHF-containing preparations with MWexceeding 70 kDa exhibited long circulation in vivo, without significantRES uptake. The blood half-life of unmodified 500 kDa PMF was found tobe more then 24 hours.

At 1:1 polymer:protein ratio, two conjugation techniques gave thehighest coupling yields (95-98% by protein): the one-step methodutilizing aminooxy-NHS coupling reagent VIII, and polymer succinylationfollowed by EDC mediated acylation. Protein adduct formation wasaccompanied with, on average, 1.5 to 2.0 fold increase in thenumber-average molecular weight (Mn). This can be an indication ofcrosslinking as the result of protein coupling with more than onepolymer chain, which can be expected considering the reactionconditions. The conjugation process caused no noticeable effect onenzymatic activity of the conjugated proteins; from 85% to 95% of theoriginal activity of native enzymes (synthetic substrates) was preservedin the conjugates. Other conjugation methods tested, such as reductiveamination or coupling with aminooxy-maleimido coupling reagent VII,showed lower yields with respect to the model protein (trypsin), butmight provide efficient coupling routes for other proteins.

The high site specificity of aminooxy reagents, mild coupling conditions(pH range 3-6), and high reaction rate (reaction half time less then 10minutes for unprotected oximes at pH 5.5) all show that this group ofcompounds has a significant potential for preparation of proteinconjugates. As expected, of the two crosslinker types tested the oximelinked conjugate showed higher degradation rate.

Animal data demonstrated PHF feasibility for designing long-circulatingprotein conjugates. Basic biokinetics data were obtained for two modelPHF-Trypsin conjugates with Mn of ˜250 kDa and ˜350 kDa (SEC estimatedparticle size of approximately 14 nm and 15 nm and protein load of 10%and 25%, respectively). A 70-fold increase in the main fraction bloodhalf-life was observed for both PHF-Trypsin conjugates, as compared tothe unmodified trypsin (8 hours vs. 7 minutes), with reduced hepatic andrenal uptake and no noticeable organ-specific accumulation. This data,notably obtained for unoptimized and unfractionated conjugates, iscomparable with the average 35-fold blood half-life prolongationreported for circulation of PEG conjugates in rodents (see, for example,(1) Delgado C, Francis G, Fisher D. (1992) The uses and properties ofPEG-linked proteins. Crit. Rev. in Ther. Drug Carrier Syst. 9, 249-304;and (2) Nucci M, Shorr R, Abuchovski A. (1991) The therapeutic value ofpoly(ethylene glycol) modified proteins. Adv. Drug Del. Rev. 6,133-151).

The results suggest that both the oxime and ester modified polyals arefeasible for preparation of fully functional biodegradable proteinconjugates. The reversible, pH-sensitive character of the oxime linkagecan be especially useful when pH dependent (e.g., lysosomal) drugrelease is desired.

Thus, all aspects of the obtained data suggest that PHF (and, likely,other semi-synthetic and fully synthetic hydrophilic polyals; See, forexample, M. Yin et al. Fully synthetic hydrophilic polyacetals. Underreview (2002)) have a significant potential as a platform for proteinmodification. In light of the present disclosure, PHF can be considereda viable and potentially superior biodegradable replacement forpolyethylene glycol, especially in applications requiring chronic and/orhigh dose administration. The dependence of conjugate stability on thedegree of PHF chain modification, protein load, crosslinker length andstructure, as well as long term hydrolytic stability of the conjugates,is a subject of ongoing research. The work is being extended toprototype conjugates of clinically relevant proteins.

Bioconjugates comprising hydrophilic, essentially fully degradablepolyacetal modules and model enzymes were successfully prepared withhigh yields and preservation of activity, utilizing conjugationstrategies based on acylation as well as reductive and non-reductiveamination. New aminooxy-reagents enabling one step protein modificationwere developed to address the problem of preparation of essentiallyfully biodegradable bioconjugates. The data suggests several potentialapplications for both essentially fully degradable hydrophilicpolyacetals and aminooxyreagents (e.g., bifunctional reagents of theinvention) in protein modification, in particular where chronic or highdose administration is required.

Example 4 Hydrophilic Polyals: Biomimetic Biodegradable StealthMaterials for Pharmacology and Bioengineering

As discussed above, acyclic hydrophilic polyals can be prepared viaeither polymerization of suitable monomers, or lateral cleavage ofcyclic polyals (e.g., polysaccharides). Both fully synthetic andsemi-synthetic polyals of various types were prepared and characterizedin vitro and in vivo as model structural and interface components ofbioconjugates, nanoparticles and other macromolecular and supramolecularconstructs.

Experimental

Semi-Synthetic Polyals. In certain embodiments, semi-Synthetic Polyalswere prepared from polyaldoses and polyketoses via complete lateralcleavage of carbohydrate rings with periodate in aqueous solutions, withsubsequent conversion of aldehyde groups into hydrophilic or otherpharmaceutically useful moieties, e.g. via borohydride reduction:

Fully Synthetic Polyals. Condensation of vinyl ethers with protectedsubstituted diols was found to be an affective method for hydrophilicpolyal formation. Efficacy of other methods, such as cycle openingpolymerization, depended on the degree of substitution and bulkiness ofthe protective groups.

Solvent systems, catalysts and other factors were optimized to obtainhigh molecular weight products.

Model Derivatives. Synthetic and semisynthetic polyals were derivatizedthrough either terminal or pendant functional groups to obtain modeldrug carriers and bioconjugates of various types.

Protected fully synthetic polyal with vinyl terminal groups (a productof condensation of mono-Fmoc-tris(hydroxymethyl)methane and ethyleneglycol divinyl ether) was grafted to modified controlled pore glass,terminally modified with 1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol(PTE), then deprotected and cleaved from the support.

Semi-synthetic poly-(hydroxymethylethylene hydroxymethyl-formal) (PHF)was modified through the terminal vicinal glycol group present on one ofthe termini via periodate oxidation with either (a) subsequent reductiveamination with 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (PEA) or(b) non-reductive amination with aminooxyacetic acid.

Pendant hydroxyl groups in both synthetic and semi-synthetic polyalswere modified via direct acylation or alkylation (e.g., with succinicanhydride or epichlorohydrine). Alternatively, semi-synthetic polymerswith pendant 1,2-glycol groups were produced and derivatized throughperiodate oxidation followed by either reductive or non-reductiveamination.

Terminal derivatives were further used to obtain polyal-modifiedliposomes, polyal-modified proteins and graft copolymers (modellong-circulating nanocarriers). Pendant-modified polyal derivatives wereused as structural backbones in various small molecule and proteinconjugates.

Radiolabeling. For biokinetics studies, polyals, their derivatives andconjugates were labeled with ¹¹¹In. Trace amounts of chelatingdiethylene triamine pentaacetate (DTPA) groups were introduced by directacylation with DTPA dicycloanhydride. DTPA derivatives were labeled with¹¹¹In by transchelation from [¹¹¹In] indium citrate at pH=5.5 andpurified by size exclusion HPLC.

In Vitro Characterization. Proton and ¹³C NMR were performed tocharacterize the structures of the obtained polyals and theirderivatives. Hydrodynamic sizes were determined by size exclusion HPLCand, where appropriate, photon correlation light scattering. Hydrolyticdepolymerization was studied in unbuffered and phosphate bufferedaqueous media as a function of pH. Solubilities were studied in waterand commonly used organic solvents. Activities of model polyal-modifiedenzymes (e.g., trypsin) were studied using synthetic substrates.Interaction of PHF with antibodies specific to the PHF precursor(dextran B-512) were studied by size exclusion HPLC and (usingfluorophore-labeled antibodies) by fluorescence polarization lifetimeinvestigation.

Cell Culture Models. Activities of clinically relevant model conjugatesof interferon α (IFN) were studied in IFN-sensitive cell cultures (TF-1,A375). Binding of formylpeptide conjugates to white blood cells wasperformed using white blood cells isolated from rodent blood andconjugates co-labeled with a fluorophore (FITC).

Animal Models. Acute toxicity of the lead polyal (PHF) was studied inoutbred mice at 0.1 mg/kg to 4 g/kg. Large molecular weight (>160 kDa)polymer preparations were used to avoid renal clearance that would maskthe possible toxic effects.

Biokinetics and biodistributions were studied by blood and tissuesampling in rats, mice and rabbits using ¹¹¹In labeled prepartations atclinically relevant doses. Fast initial stages (if present) were studiedby dynamic gamma-scintigraphy in anesthetized animals.

Biokinetics of unmodified polyals of various molecular weights werestudied in normal mice, rats and rabbits. Biokinetics of modelnanocarriers (PHF graft copolymers) and polyal-modified liposomes werestudied in rats. Biokinetics of formylpeptide conjugates were studied innormal rabbits and rabbits with focal bacterial inflammation. Proteinconjugates were studied in mice and rats.

Toxicities and biological activities of model conjugates (IFN, G-CSF,antineoplastic small molecules) were studied in mice, using relevantmodels (Cytoxan challenge for G-CSF; cancer xenografts in nude mice forantineoplastic preparations)

Synthesis and characterization. In the semi-synthetic polyal syntheses,yields and molecular weights of the products were found to varydepending on the precursor polysaccharide. Polyals from polysaccharideprecursors of high regularity and low branching, such as dextran B-512and inulin, were obtained practically without depolymerization and withnearly theoretical yields. SEC HPLC elution profiles of the productspractically reproduced the profiles of the respective precursorpolysaccharides (e.g., from 3 kDa to 1,500 kDa for PHF). DextranB-512-specific antibodies did not bind PHF.

In the vinyl ether condensation-based syntheses, several protected diolderivatives were tested and found to be equally effective at thepolymerization stage. However, Fmoc protection was found to enablebetter yields at the subsequent derivatization and deprotection stages.Fully synthetic polyals with 5-15 kDa main fraction were produced withhigh yields. Large molecular weight preparations (>35 kDa) were isolatedby fractionation.

All obtained polymers were examined by ¹³C and ¹H NMR, and were found tobe in agreement with the expected fully acyclic polyal structures.Polyals showed the expected profile of hydrolytic degradation, beingessentially stable at pH from 7 to ca. 10.5, and hydrolyzable atlysosomal pH of 5. Large molecular weight polyals (MW>50 kDa) weresoluble in water and several organic solvents (e.g. DMF, DMSO, Py).Lower MW fractions were also soluble in methanol and Py/methanol andother solvents and mixtures.

Polyals tested to date in animals were found to be essentiallynon-toxic. The most extensively studied PHF showed no signs of toxicityand no weight loss even after intravenous administration of 4 gram perkg of body weight.

Biokinetics and biodistribution of ¹¹¹In labeled PHF was studied inrats. Low molecular weight fractions were found to be rapidly excretedthrough kidneys without significant accumulation in any tissue. For a 50kDa preparation, blood half-life was 2 hours with label content intissues below 0.05% injected dose/g (24 hours post injection). Highmolecular weight fractions (e.g., 500 kDa) had long blood half-lives (25hours), with no preferential accumulation in any tissue (generally,below 0.2% dose/g at 72 hours). Hepatic, splenic and lymphaticaccumulation levels were not much higher than in other tissues (0.4±0.1%dose/g), suggesting spontaneous liquid phase endocytosis, rather thanphagocyte recognition, as the main uptake mechanism.

Derivatives. Terminal and pendant group derivatives were successfullyprepared for in vitro and in vivo characterization.

Protein conjugates. Succinyl-PHF was used to prepare model proteinconjugates (trypsin, IFN, G-CSF) with protrein:polymer ratio from ca.1:1 to 1:2 with nearly theoretical yields (by protein). The conjugatesshowed the expected increase in hydrodynamic diameter (to ca. 10-11 nm)and insignificant (0%-5%) loss of specific activity, as measured invitro or in cell culture models. All protein conjugates showed dramaticimprovements in biokinetics, e.g., blood half-life increases from 7 and11 minutes to 8 and 13 hours (trypsin and IFN, respectively), and a 5-10fold reduction in renal accumulation upon both IV and SC administration.The G-CSF conjugate was found to retain the in vivo activity, and had apotentially superior activity dynamics. The initial data obtained withmodel hemoglobin and other conjugates suggested that protein-polyalconjugates, unlike analogous conjugates of PEG, caused a much lesssignificant or no renal vacuolization, probably depending on the doseand timeframe (work in progress).

Small Peptide. Formylpeptide N-formyl-Met-Leu-Phe-Lys) conjugates werefound to have high affinity to white blood cells (via formylpeptidereceptor). Administration of such (labeled) conjugates in rats andrabbits resulted in efficient in vivo labeling of white blood cells andtheir invasions in focal bacterial inflammations. While inflammationlabeling efficacy was equal to that of unmodified formylpeptide, renalaccumulation (and, respectively, radiation does) was reduced by 81-88%,depending on the molecular weight, and hepatic and splenic accumulationswere reduced by 40% for low molecular weight (15 kDa) preparations.

Liposomes. Polyal-modified 100 nm DPPC/Cholesterol liposomes showed asignificantly prolonged circulation upon IV administration in rats vs.unmodified liposomes, e.g., 50% clearance during 30 min. vs. 90%clearance during 15 min., respectively. Work is in progress to optimizepolyal content and molecular weight in order to obtain fullybiodegradable long-circulating liposomes.

Drug carriers. Model sterically protected nanocarriers (hydrodynamicdiameter 16±4 nm) assembled using 20 kDa poly-L-lysine as a backbone and10 kDa PHF as protective graft showed strong correlation of bloodhalf-life (rat) with the number of PHF chains per backbone. Whileunprotected polylysine has blood half-life of ca. 20 seconds,PHF-modified carriers with 10 and 20 graft molecules per backbone hadhalf-lives of 9.8 and 25.3 hours, respectively.

Small molecule conjugates. Being strongly hydrophilic (in some caseseven hygroscopic) polymers, polyals are suitable for solubilizingstrongly hydrophobic small molecules. Several small molecule conjugateswere prepared through either direct acylation of polyals (DTPAcycloanhydride, succinic anhydride) or alkylation (epichlorohydrine), orthrough non-reductive amination using bifunctional aminooxy-reagentsdeveloped in our laboratories (H₂N—O—R—X, where X is a functional group,e.g., N-maleimide or N-hydroxysuccinimide ester). Conjugates of modelantineoplastic drugs were soluble at drug content from at least ca. 5-7%w/w (most hydrophobic substances) to ca. 15% (anthracyclines, such asdoxorubicine). One of such conjugates was tested in mouse xenograftmodels (LS174t and HT26 in nude mice) and demonstrated lower toxicityand higher antineoplastic activity than the respective unmodified drug.For example, animal survival 55 days post treatment start was 80% in theconjugate-treated group vs. 40% in the group treated with unmodifieddrug (same dose), and 20% in the untreated control group. Optimizationof conjugate size, composition and conjugation/release chemistry is asubject of our ongoing work.

One goal of this study was to determine whether a macromolecularmaterial built of the common acyclic structures of carbohydrates wouldhave the set of features necessary for advanced pharmacologicalengineering. These include “inertness” in vivo (non-bioadhesiveness, or“stealth” properties), biodegradability of the main chain, low toxicity,and technological flexibility.

The polyal main chain was found to be stable at physiological conditions(pH=7 and above) but sensitive to proton-catalyzed hydrolysis at pH<7.Low pH is characteristic for the intracellular lysosomal and caveolarcompartments. Therefore, cellular uptake of polyal-based preparationscan be expected to result in complete non-enzymatic hydrolysis of themain chain at a moderate rate. The constitutive units of polyals havelow toxicity and can be metabolized via major metabolic pathways and/orexcreted. As compared to hydrolysis-resistant polymers, e.g.,polyethyleneglycol, this appears to be a significant advantage,especially in preparations intended for high dose or chronicadministration, where long-term cell vacuolization, intracellularpolymer storage, and the associated functional abnormalities can resultin significant safety risks.

For particles and large macromolecules, blood half-life is amathematically exact measure of the overall polymer reactivity [PapisovM. I., Adv. Drug Delivery Rev., 1995, 16:127-137]. The results of invivo evaluation of the lead polyal (PHF) showed that neither linear norhighly branched derivatives were recognized by reticuloendothelialcells. The obtained biokinetics data provides a clear evidence that thecentral goal of this study, developing of biodegradable materials withminimized interactions with biological milieu, have been achieved.

Hydrophilic polyals of various structures have been successfullysynthesized. These polymers demonstrated excellent technologicalflexibility and biological properties. The obtained data suggestsseveral potential applications for these hydrophilic, essentially fullybiodegradable polymers, in particular in advanced drug delivery systems,protein and small molecule modification, and drug carrier engineering.

1. A conjugate comprising a carrier substituted with one or moreoccurrences of a moiety having the structure:

wherein each occurrence of M is independently a pharmaceutically usefulmodifier; the carrier comprises a biodegradable biocompatible polymerselected from polyacetals or polyketals and the molecular weight of thecarrier is between about 0.5 and about 1500 kDa; wherein at least asubset of the polyacetal repeat structural units have the followingchemical structure:

wherein for each occurrence of the n bracketed structure, one of R¹ andR² is hydrogen, and the other is a biocompatible group and includes acarbon atom covalently attached to C¹; R^(x) includes a carbon atomcovalently attached to C²; n is an integer; each occurrence of R³, R⁴,R⁵ and R⁶ is a biocompatible group and is independently hydrogen or anorganic moiety; and for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl groupsuitable for oxime formation; wherein at least a subset of the polyketalrepeat structural units have the following chemical structure:

wherein each occurrence of R^(1a) and R^(2a) is a biocompatible groupand includes a carbon atom covalently attached to C¹, and at least oneof R^(1a), R^(2a), R³, R⁴, R⁵ and R⁶ comprises a carbonyl group suitablefor oxime formation and each occurrence of L^(M) is independently anoxime-containing linker.
 2. The conjugate of claim 1, wherein eachoccurrence of L^(M) is independently a moiety having the structure:

wherein each occurrence of L^(M1) is independently a substituted orunsubstituted, cyclic or acyclic, linear or branched C₀₋₁₂alkylidene orC₀₋₁₂alkenylidene moiety wherein up to two non-adjacent methylene unitsare independently optionally replaced by CO, CO₂, COCO, CONR^(Z1),OCONR^(Z1), NR^(Z1)NR^(Z2), NR^(Z1)NR^(Z2)CO, NR^(Z1)CO, NR^(Z1)CO₂,NR^(Z1)CONR^(Z2), SO, SO₂, NR^(Z1)SO₂, SO₂NR^(Z1), NR^(Z1)SO₂NR^(Z2), O,S, or NR^(Z1) wherein each occurrence of R^(Z1) and R^(Z2) isindependently hydrogen, alkyl, heteroalkyl, aryl, heteroaryl or acyl. 3.The conjugate of claim 2, wherein one or more occurrences of L^(M1)independently comprises a maleimide-containing crosslinker.
 4. Theconjugate of claim 3, wherein one or more occurrences of L^(M1)independently comprises a4-(N-maleimidomethyl)cyclohexane-1-carboxylate, m-maleimidobenzoyl or a4-(p-maleimidophenyl)butyrate crosslinker.
 5. The conjugate of claim 1,wherein one or more occurrences of M comprises, or is attached to thecarrier through, a biodegradable bond.
 6. The conjugate of claim 4,wherein the biodegradable bond is selected from the group consisting ofacetal, ketal, amide, ester, thioester, enamine, imine, imide, dithio,and phosphoester bond.
 7. The conjugate of claim 1, wherein the carrieris a biodegradable biocompatible polyacetal wherein at least a subset ofthe polyacetal repeat structural units have the following chemicalstructure:

wherein for each occurrence of the n bracketed structure, one of R¹ andR² is hydrogen, and the other is a biocompatible group and includes acarbon atom covalently attached to C¹; R^(x) includes a carbon atomcovalently attached to C²; n is an integer; each occurrence of R³, R⁴,R⁵ and R⁶ is a biocompatible group and is independently hydrogen or anorganic moiety; and for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl groupsuitable for oxime formation.
 8. The conjugate of claim 1, wherein thecarrier is a biodegradable biocompatible polyketal wherein at least asubset of the polyketal repeat structural units have the followingchemical structure:

wherein each occurrence of R^(1a), and R^(2a) is a biocompatible groupand includes a carbon atom covalently attached to C¹; R^(x) includes acarbon atom covalently attached to C²; n is an integer; each occurrenceof R³, R⁴, R⁵ and R⁶ is a biocompatible group and is independentlyhydrogen or an organic moiety; and for each occurrence of the bracketedstructure n, at least one of R^(1a), R^(2a), R³, R⁴, R⁵ and R⁶ comprisesa carbonyl group suitable for oxime formation.
 9. The conjugate of claim8, wherein one or more occurrence of M is selected from the groupconsisting of proteins, antibodies, antibody fragments, peptides,antineoplastic drugs, hormones, cytokines, enzymes, enzyme substrates,receptor ligands, lipids, nucleotides, nucleosides, metal complexes,cations, anions, amines, heterocycles, heterocyclic amines, aromaticgroups, aliphatic groups, intercalators, antibiotics, antigens,immunomodulators, and antiviral compounds.
 10. The conjugate of claim 1,wherein the conjugate is water-soluble.
 11. The conjugate of claim 1,wherein the conjugate comprises a pharmaceutically useful modifier and adetectable label.
 12. A composition comprising the conjugate of claim 1and a pharmaceutically suitable carrier or diluent.
 13. A compositioncomprising a conjugate of claim 1 associated with an effective amount ofa therapeutic agent; wherein the therapeutic agent is incorporated intoand released from said conjugate matrix by degradation of the conjugatematrix or diffusion of the agent out of the matrix over a period oftime.
 14. The composition of claim 13 wherein said conjugate is furtherassociated with a diagnostic label.
 15. The conjugate of claim 4,wherein one or more occurrences of L^(M1) independently comprises a4-(N-maleimidomethyl)cyclohexane-1-carboxylate crosslinker.
 16. Theconjugate of claim 4, wherein one or more occurrences of L^(M1)independently comprises a m-maleimidobenzoyl crosslinker.
 17. Theconjugate of claim 4, wherein one or more occurrences of L^(M1)independently comprises a 4-(p-maleimidophenyl)butyrate crosslinker. 18.The conjugate of claim 1, wherein the molecular weight of the carrier isbetween about 1 and about 1000 kDa.
 19. The conjugate of claim 7,wherein the molecular weight of the carrier is between about 1 and about1000 kDa.
 20. The conjugate of claim 8, wherein the molecular weight ofthe carrier is between about 1 and about 1000 kDa.
 21. The conjugate ofclaim 1, wherein the carrier is hydrophilic.
 22. The conjugate of claim7, wherein the carrier is hydrophilic.
 23. The conjugate of claim 8,wherein the carrier is hydrophilic.